Human Erythroleukemia Cells Research Articles

Effect of Sch 55700, a humanized monoclonal antibody to human interleukin-5, on eosinophilic responses and bronchial hyperreactivity.

This report describes the development and the biology of Sch 55700, a humanized monoclonal antibody to human IL-5 (hIL-5). Sch 55700 was synthesized using CDR (complementarity determining regions) grafting technology by incorporating the antigen recognition sites for hIL-5 onto consensus regions of a human IgG4 framework. In vitro, Sch 55700 displays high affinity (Kd = 20 pmol/l) binding to hIL-5, inhibits the binding of hIL-5 to Ba/F3 cells (IC50 = 0.5 nmol/l) and blocks IL-5 mediated proliferation of human erythroleukemic TF-1 cells. In allergic mice, Sch 55700 (0.1-10 mg/kg, i.p. or i.m.) inhibits the influx of eosinophils in the lungs, demonstrates long duration of activity and the anti-inflammatory activity of this compound is additive with oral prednisolone. In allergic guinea pigs, Sch 55700 (0.03-30 mg/kg i.p.) inhibits both the pulmonary eosinophilia and airway hyperresponsiveness and at 30 mg/kg, i.p. inhibited allergic, but not histamine-induced bronchoconstriction. In allergic rabbits, Sch 55700 blocks cutaneous eosinophilia. Sch 55700 (0.1-1 mg/kg i.p.) also blocks the pulmonary eosinophilia and neutrophilia caused by tracheal injection of hIL-5 in guinea pigs. In allergic cynomolgus monkeys, a single dose of Sch 55700 (0.3 mg/kg i.v.) blocks the pulmonary eosinophilia caused by antigen challenge for up to six months. Sch 55700 is, therefore, a potent antibody against IL-5 in vitro and in a variety of species in vivo that could be used to establish the role of IL-5 in human eosinophilic diseases such as asthma.

Effects and mechanisms of total flavonoids of Astragali Radix and calycosin on inhibiting human erythroleukemia cell line K562

To investigate the effects of total flavonoids of Astragali Radix (TFA) and calycosin on apoptosis induction and cell cycle in human erythroleukemia cell line K562. MTT assay was used to measure the inhibition effect on the proliferation of K562 cells cultured with TFA and calycosin. The effect of TFA and calycosin on cell cycle in K562 was detected by PI staining. The apoptosis induction effect was measured by Annexin V/PI double staining. RT-PCR was used to determine the level of Cyclin D1 mRNA in K562 cells after treated with TFA and calycosin. TFA and calycosin could inhibit the proliferation of K562 cells, the 50% inhibiting concentration of TFA and calycosin were 98.63 mg x L(-1) and 130.32 mg x L(-1) respectively. TFA and calycosin could not induce apoptosis in K562 cells, but could increase the number of cells in the G0/G1 phase. The level of Cyclin D1 mRNA in K562 cells decreased after treated with TFA and calycosin. TFA could inhibit the proliferation of K562 cells, and attribute to arrest them in the G0/G1 phase and decrease Cyclin D1 mRNA.

Induction of apoptosis and cell cycle arrest by Bis (2-ethylhexyl) phthalate produced by marine Bacillus pumilus MB 40

Marine microorganisms represent a potential source for the production of biomedically useful compounds active against inflammation, cancer, diabetes, etc. Marine Bacillus pumilus MB 40 (GenBank accession no. HQ705771) isolated from deep sea water column (1000m depth) near Andaman and Nicobar islands produced a bioactive lead, Bis (2-ethylhexyl) phthalate (BEHP) with a molecular formula of C6H4(CO2C8H17)2 and a molecular ion at m/z 391 (M+). Anti proliferative effect of the isolated compound was examined by MTT assay in human erythroleukemic K562 cells and the IC50 of BEHP was found to be 21μM. BEHP was able to induce apoptosis involving caspases pathway, besides regulating mitochondrial enzymes. Further, western blot analysis revealed the activation of caspases family proteins viz., caspase 8, caspase-9 and caspase-3. An increase in the expression of Bax mRNA concomitant with a decrease in mRNA of Bcl-2 in BEHP treated K562 cells was also observed. AO/EB staining of BEHP treated K562 cells further confirmed the progression of apoptosis as evidenced by morphological changes including nuclear condensation, cell shrinkage, and formation of apoptotic bodies. Treatment of K562 cells with BEHP induced the progressive accumulation of fragmented DNA in a time dependent manner. This pattern appeared as a typical laddering distribution of DNA fragmentation due to intranucleosomal cleavage associated with apoptosis. Based on flow cytometric analysis it has become evident that the compound was also effective in arresting the cell cycle at a sub G0/G1 phase.

Constitutive expression of a Mg2+-inhibited K+ current and a TRPM7-like current in human erythroleukemia cells

Whole cell patch-clamp experiments were undertaken to define the basal K(+) conductance(s) in human erythroleukemia cells and its contribution to the setting of resting membrane potential. Experiments revealed a non-voltage-activated, noninactivating K(+) current. The magnitude of the current recorded under whole cell conditions was inhibited by an increase in free intracellular Mg(2+) concentration. Activation or inactivation of the Mg(2+)-inhibited K(+) current (MIP) was paralleled by activation or inactivation of a Mg(2+)-inhibited TRPM7-like current displaying characteristics indistinguishable from those reported for molecularly identified TRPM7 current. The MIP and TRPM7 currents were inhibited by 5-lipoxygenase inhibitors. However, inhibition of the MIP current was temporally distinct from inhibition of TRPM7 current, allowing for isolation of the MIP current. Isolation of the MIP conductance revealed a current reversing near the K(+) equilibrium potential, indicative of a highly K(+)-selective conductance. Consistent with this finding, coactivation of the nonselective cation current TRPM7 and the MIP current following dialysis with nominally Mg(2+)-free pipette solution resulted in hyperpolarized whole cell reversal potentials, consistent with an important role for the MIP current in the setting of a negative resting membrane potential. The MIP and TRPM7-like conductances were constitutively expressed under in vivo conditions of intracellular Mg(2+), as judged by their initial detection and subsequent inactivation following dialysis with a pipette solution containing 5 mM free Mg(2+). The MIP current was blocked in a voltage-dependent fashion by extracellular Cs(+) and, to a lesser degree, by Ba(2+) and was blocked by extracellular La(3+) and 2-aminoethoxydiphenyl borate. MIP currents were unaffected by blockers of ATP-sensitive K(+) channels, human ether-à-go-go-related gene current, and intermediate-conductance Ca(2+)-activated K(+) channels. In addition, the MIP current displayed characteristics distinct from conventional inwardly rectifying K(+) channels. A similar current was detected in the leukemic cell line CHRF-288-11, consistent with this current being more generally expressed in cells of leukemic origin.

A p210 BCR/ABL Mutant That Lacks Guanine Nucleotide Exchange Factor Activity Induces Erythroleukemia in a Murine Bone Marrow Transplantation Model,

Abstract 3756The p210 BCR/ABL and p190 BCR/ABL fusion proteins are constitutively active tyrosine kinases that are associated with chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) respectively. Whereas both of these fusion proteins contain equivalent contributions from ABL, they differ in the amount of BCR sequence at the NH2-terminus. We have identified a domain with guanine nucleotide exchange factor (GEF) activity which is contained within the BCR sequences of p210 BCR/ABL that distinguishes it from p190 BCR/ABL. This activity is subject to autoinhibition in the context of BCR, but is constitutively active in p210 BCR/ABL, and cannot be blocked using the tyrosine-kinase inhibitor, Imatinib mesylate. The activities of small GTPases are regulated by GEFs, and are essential for the regulation of hematopoietic stem cell differentiation and proliferation. In a human Ph-positive erythroleukemia cell line (K562), inhibiting p210 BCR/ABL-mediated Ras activation induces erythroid differentiation, while abnormal activation of Cdc42 reduces erythroid progenitors and erythroid colony-forming units in bone marrow. In order to directly investigate the contribution of the GEF activity to p210 BCR/ABL-mediated leukemogenesis, we have introduced a point mutation into p210 BCR/ABL (p210 BCR/ABL(S509A)) that eliminates the activity. The mutation has no discernable effect on tyrosine kinase activity, which is consistent with previous data that the two activities are functionally independent. We have compared p190 BCR/ABL, p210 BCR/ABL, and p210 BCR/ABL(S509A) in a murine bone marrow transplantation (BMT) model. Although all transplanted mice develop splenomegaly with leukocytosis, the p190 BCR/ABL and p210 BCR/ABL(S509A) transplanted mice exhibit a more rapid onset of disease than mice transplanted with p210 BCR/ABL (15–18 days vs 23–38 days). This difference is observed regardless of whether the donor mice are pre-treated with 5-Fluorouracil. The more rapid onset of disease induced by p190 BCR/ABL and p210 BCR/ABL(S509A) is associated with reticulocytosis, and increased numbers of nucleated red blood cells (nRBC), but no anemia. The nRBC/100 WBC counts are 0, 4.75±2.21, 277.5±190.6, and 119.5±66.0 for MIG, p210 BCR/ABL, p190 BCR/ABL and p210 BCR/ABL (S509A) respectively, thus suggesting leukemic expansion of the erythroid lineage. To confirm this difference in lineage expansion, clonogenicity assays were performed. Consistent with previous reports, p210 BCR/ABL supports the growth of CFU G/M, but not BFU-E, and colony growth is completely inhibited by Nilotinib. In contrast, p190 BCR/ABL and p210 BCR/ABL(S509A) support the growth of BFU-E (but not CFU G/M), and these colonies are completely insensitive to Nilotinib treatment. In summary, our observations suggest that a point mutation that eliminates the GEF activity of p210 BCR/ABL produces a phenocopy of p190 BCR/ABL, without affecting the tyrosine kinase activity. These results support a model in which the GEF activity that distinguishes p210 BCR/ABL from p190 BCR/ABL actively regulates disease progression by determining lineage-specific leukemic expansion. Disclosures:No relevant conflicts of interest to declare.

CoCoA/CCAR1 Pair-Mediated Recruitment of Mediator Complex Indicates Novel Pathway for the Function of GATA1 in Erythroid Differentiation

Abstract 1302The mammalian multi-protein complex Mediator, originally identified by ourselves as a nuclear receptor-specific coactivator complex, is a phylogenetically-conserved subcomplex of the RNA polymerase II holoenzyme and serves as an end-point integrator of diverse intracellular signals and transcriptional activators. The 220-kDa Mediator subunit MED1 is a specific coactivator not only for nuclear receptors but for GATA family activators, and serves as a GATA1-specific coactivator that is essential for optimal GATA1-mediated erythropoiesis. In this study, we show a novel nuclear signaling pathway for MED1 action in GATA1-induced transcriptional activation during erythroid differentiation. First, we identified the amino acid residues 681–715 of human MED1 (MED1(aa.681-715)) to be responsible for the direct interaction with GATA1. When MED1 in K562 human erythroleukemic cells was knocked down during hemin-induced erythroid differentiation, the erythroid differentiation was significantly attenuated as assessed by an erythroid differentiation score defined by the number of cells positive for benzidine staining, and the expressions of the GATA1-targeted and erythroid differentiation marker genes, β-globin, γ-globin, PBGD and ALAS-E, were prominently attenuated. However, overexpressions of the N-terminal MED1 truncations without and with nuclear receptor recognition motifs, MED1(aa.1–602) and MED1(aa.1–703), respectively, but neither of which could bind to GATA1 (above), prominently enhanced erythroid differentiation of K562 cells. Luciferase reporter assays by using the human γ-globin promoter and Med1−/− mouse embryonic fibroblasts (MEFs) showed that these N-terminal MED1 truncations rescued GATA1-mediated transactivation, indicating that MED1(a.a.1–602) served as the functional interaction surface for GATA1. Hence, a putative bypass for GATA1-MED1 pathway appears to exist, and is expected to interact with the N-terminus of MED1. As a candidate bypass system, we tested both the recently reported bypass molecule for a nuclear post-activator signaling, CCAR1, and its partner coactivator CoCoA. CCAR1 was reported by others to bypass the estrogen receptor-mediated transactivation by a simultaneous binding of CCAR1 with the estrogen receptor and the N-terminus of MED1. Functionally, serial luciferase reporter assays by using the γ-globin promoter and MEFs demonstrated cooperative transactivation by combinations of GATA1, CCAR1, CoCoA and/or the N-terminus of MED1, but the transactivation mediated by the N-terminus of MED1 was not as prominent as the one mediated by the full-length MED1. An overexperssion of CCAR1 or CoCoA in K562 cells prominently enhanced both the GATA1-mediated erythroid differentiation and the expressions of the GATA1-targeted genes. Next, the mechanisms underlying the CCAR1- and CoCoA-mediated GATA1 functions were analyzed by serial GST-pulldown and mammalian two-hybrid assays, and the following results were obtained. (i) The N-terminus of CCAR1 interacted with the C-terminus of CoCoA. (ii) The N-terminus of MED1 interacted with both the N- and C-termini of CCAR1. (iii) While the N-terminal zinc-finger domain of human GATA1 (GATA1(a.a.204–228)) is known to bind to the well-known GATA1 partner FOG1, intriguingly, the C-terminal zinc-finger domain of GATA1 (GATA1(a.a.258–272)) interacted with all three of the following cofactors; MED1 (MED1(aa.681–715)), CCAR1 (at the C-terminus) and CoCoA (at both the N- and C-termini). The affinity of CoCoA to bind to GATA1 appeared to be a little higher than the other. Thus, the GATA1(a.a.258-272) zinc finger appears to serve as a docking surface for multiple coactivating proteins, where both MED1 and CoCoA/CCAR1 pair can interact, probably in a competitive manner, or perhaps simultaneously. Here, both CoCoA/CCAR1 as a pair and CCAR1 by itself can serve as a bypass. Finally, ChIP assays of hemin-treated K562 cells showed that GATA1, CCAR1/CoCoA and MED1 were all recruited onto the γ-globin promoter during transactivation. Taken together, besides a direct interaction between GATA1 and MED1, the CoCoA/CCAR1 pair appears to relay the GATA1 signal to MED1. The multiple modes of mechanisms for transcription mediated by the GATA1-MED1 axis might contribute to a fine tuning of the GATA1 function, not only during erythropoiesis but also in other GATA1-mediated homeostasis events, within a living animal. Disclosures:No relevant conflicts of interest to declare.

JAK2 V617F, PRMT5, and GATA-1 Form a Regulatory Loop That Controls Autophagy Via Effects on ATG3 Gene Expression

Abstract 2822The JAK2 V617F mutation is the most prevalent genetic abnormality in Philadelphia chromosome negative myeloproliferative neoplasia. We recently discovered that JAK2 V617F phosphorylates and inactivates PRMT5, a type II arginine methyltransferase that promotes transcriptional repression. To evaluate the PRMT5 dependent and independent effects of JAK2 V617F, we performed microarray analysis of human erythroleukemia cells (homozygous for JAK2 V617F) treated with a JAK2 inhibitor or transduced with PRMT5 shRNA. We discovered 5 autophagy-related genes (ATG3, ATG7, ATG2B, ATG5, and ATG16L1) that were positively regulated by JAK2 V617F, but negatively regulated by PRMT5. PRMT5 was bound to the promoters of these genes and binding was enhanced when PRMT5 was re-activated by a JAK2 inhibitor. Sequence analysis of ATG promoters identified at least one potential GATA-1 binding site in each ATG gene. Yet, knock-down of GATA-1 decreased the expression of ATG3 (by approximately 50%), but not the other ATG genes. ChIP analysis identified binding of both GATA-1 and PRMT5 to the ATG3 promoter, which suggests that ATG3 is transcriptionally induced by GATA-1 when PRMT5 is inactivated by JAK2 V617F. But when PRMT5 was active, it was bound to the GATA-1 promoter and ATG3 expression was downregulated. As JAK2 V617F (but not wild type) induces ATG3 expression and autophagy in AML cells and ATG3 levels are elevated in CD34+ cells from polycythemia vera patients, we conclude that JAK2 V617F promotes autophagy by inactivating PRMT5 and promoting GATA-1-dependent transcription of ATG3. Targeting autophagy with JAK2 inhibitors or other agents may be efficacious in the treatment of myeloproliferative neoplasia. Disclosures:No relevant conflicts of interest to declare.

In Vitro Characterization of R14W Mutation in SEC23B, the CDAII Causative Gene

Abstract 2098Congenital dyserythropoietic anemias (CDAs) designate a group of genetic disorders in which ineffective erythropoiesis is the predominant mechanism of anemia marked by distinct morphologic abnormalities of the majority of erythroblasts in the bone marrow. CDA type II (CDAII) is the most common type of CDA. It is characterized by a recessive model of inheritance, mild to moderate anemia, jaundice, and splenomegaly (Fukuda MN, Glycobiology 1990; Fukuda MN, Clin Haematol 1993), by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance (Schwarz K and Iolascon A. et al., Nat Genet. 2009). The specific hallmark of diagnosis is the presence of the more abundant protein of membrane red cell, band 3, in a hypoglycosilated state; this is thinner and migrated slightly faster than in controls on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Anselstetter V et al., Br J Haematol 1977).The causative gene of CDAII, SEC23B, is a member of the SEC23 subfamily of the SEC23/SEC24 family, which is involved in vesicle trafficking. The encoded protein has similarity to yeast Sec23p (66.4%) component of COPII, the coat protein complex responsible for vesicle budding from the ER. The function of this gene product has been implicated in cargo selection and concentration.The SEC23B gene spans approximately 54 kb on human chromosome 20p11.23 and codifies for a protein of 767 aminoacids divided in five functional domains: zink finger, trunk, β-sheet, helical and gelsolin domain. Although most of the SEC23B mutations are sporadic events, 4 mutations (R14W, E109K, R497C, I318T) accounted for more than 50% of mutated alleles. The aim of this study is the in vitro characterization of the R14W mutation, the most frequent variant in Italy, particularly in South of Italy (Russo R et al., Am J Hematol. in press). We used the human erythroleukemia cell line, HEL, as in vitro model because it is more similar to mature red cell. By using in silico tool ESyPred3D Web Server 1.0, we predicted that this aminoacidic substitution alters the zink finger domain 3D structure, when compared to wild type protein. This tool implements homology modeling approach followed by a final analysis with MODELLER release 4 in order to build a 3D model of the submitted protein (Lambert et al, 2002). However, when we transfected the SEC23B-R14W we observed a strong reduction of gene expression in the mutant when compared to SEC23B-wt construct by qRT-PCR. These results have been also confirmed at the protein level. In fact the protein expression of SEC23B-R14W showed a reduction comparable to gene expression respect to SEC23B-wt construct. Immunofluorescence analyses by confocal microscopy, were used for the investigation of the cellular localization of SEC23B-R1W protein and, interestingly, the localization of mutant protein was not changed when compared to that wt. Our data allow us to hypothesize that the mutation R14W gives rise to anomalous protein product quantity, but the protein function would be like not altered.Our findings demonstrated that the most frequent mutation found in Italy, SEC23B-R14W, results in a reduced half-life of the mutated mRNA, without altering the cellular localization in HEL cell line. SEC23B belongs to a multiproteic compelx that assembles with the others complex proteins in accordance with a specific structure. Each structure establishes a cargo selectivity. Further studies are necessary in order to understand what is the role of SEC23B in selectivity of the cargo in erythroid cells and how its disruption could determines the appearance of the principal pathological phenotype in CDAII patients, for example the hypoglycosilation of band 3. Disclosures:No relevant conflicts of interest to declare.

Lenalidomide Upregulates Erythropoietin Receptor Expression in Hematopoietic Progenitors by Modulating Receptor Turnover

Abstract 2382The erythropoietin receptor (EpoR) is a type 1 cytokine receptor and key effector of Epo signaling. Maturation and shuttling of receptor to the plasma membrane is dependent on its association with Janus Kinase 2 (JAK2). Upon ligand binding, EpoR homo-dimerizes to activate JAK2, which in turn phosphorylates tyrosine residues on the receptor cytoplasmic tail permitting recruitment and phosphorylation of the signal transduction and activator of transcription (STAT)-5. Lenalidomide (LEN), a second generation immunomodulatory drug (IMiD), promotes the expansion of primitive erythroid progenitors in vitro and induces the expression of genes involved in erythroid lineage commitment (JCI 2008;118(1):248-58; PLoS medicine 2008;5(2):e35). In del(5q) clones, LEN is cytotoxic by inhibiting the haplodeficient serine/threonine phosphatase PP2A (Wei S, et. al PNAS 2009). To investigate the mechanism by which LEN promotes erythropoiesis, we investigated its action on EpoR cellular dynamics. Using the human erythroleukemia cell line, UT-7, treatment with LEN elicited concentration dependent upregulation of EpoR in whole cell lysates demonstrable by immunoblot (IB) analysis at concentrations ranging from 0.1–10μM, reaching a 2-fold increase vs. controls. Treatment with pomalidomide, an IMiD with superior erythropoietic activity, displayed greater potency, increasing cellular EpoR at concentrations as low as 1nM; whereas thalidomide was ineffective. Upregulation of EpoR was apparent as early as 1 hour (h) after LEN exposure, reaching maximal receptor induction at 8h. Isolation of membrane and cystosolic fractions followed by IB showed that EpoR was upregulated predominantly in the membrane fraction, increasing 4.3-fold. IB of detergent insoluble membrane raft (MR) fractions showed that LEN promoted lipid raft assembly and F-actin polymerization accompanied by translocation of EpoR into MR microdomains with JAK2, STAT-5 and Lyn signal intermediates. Pretreatment with the Rho-GTPase inhibitor, Y27632, suppressed LEN effects on MR and F-actin. Immunoprecipitation (IP) of EpoR showed that JAK2 co-precipitated with the receptor in LEN treated cells, suggesting drug induced release of pre-assembled EpoR/JAK2 complexes from golgi and endosomal compartments. Q-PCR showed no changes in EpoR gene mRNA expression up to 24 hours after drug exposure, supporting post translational drug effects. To confirm that the observed changes in EpoR dynamics in UT-7 cells extends to normal erythroid progenitors, we assessed EpoR expression and MR assembly (cholera toxin B labeled GM1) by fluorescence microscopy in CD71+ erythroid precursors from three normal bone marrow donors. Expression of EpoR increased a mean of 17% (range:0-68%) after 1h LEN [1μM] exposure (p=0.003), accompanied by an increase in MR aggregates. Because the LEN inhibitable target PP2A is a key regulator of Rho-GTPase guided receptor trafficking and AP-1 directed formation of MR targeted clathrin-coated vesicles, we examined its effects on EpoR induction. Pretreatment of UT-7 cells with the PP2A activator FTY720 antagonized the upregulation of EpoR by LEN, whereas treatment with the PP2A inhibitor cantharadin [0.5μM] induced EpoR protein expression. Inhibition of dynamin activity, a PP2A sensitive GTPase involved in type 1 receptor recycling from membrane to endosomes, recapitulated the effects of cantharadin, inducing EpoR expression. In summary, these findings indicate that LEN modulates EpoR equilibrium, mediated in part through its inhibitory effect on PP2A. LEN induces rapid translocation of mature EpoR/JAK2 complexes to the plasma membrane while promoting the assembly of MRs with signaling competent receptor platforms to optimize cytokine signal integrity. Disclosures:List:Celgene: Consultancy.

Enantiomer-Specific Transformation by 2HG Is Linked to Opposing Effects on α-Ketoglutarate-Dependent Dioxygenases

Abstract LBA-4 Somatic mutations in IDH1 and IDH2 occur frequently in clonal myeloid disorders and result in the neomorphic ability of IDH to convert α-ketoglutarate (2-OG) to the R-enantiomer of 2-hydroxyglutarate (R-2HG) (Dang, et al Nature 462: 739, 2009). 2OG is an essential cofactor for many metabolic enzymes, including the TET family of 5-methylcytosine hydroxylases and the EglN family of prolyl-4-hydroxylases, and 2HG has been shown to inhibit several 2OG-dependent dioxygenases in vitro, including TET2 (Xu, et al Cancer Cell 19: 17, 2011; Figueroa, et al Cancer Cell 18: 1, 2010). We recently showed that the (S) enantiomer of 2HG (S-2HG), but not the (R) enantiomer of 2HG (R-2HG), inhibits the EglN prolyl-4-hydroxylases (Koivunen, et al. Submitted for publication). Moreover, we found that R-2HG can act as a cofactor to promote the hydroxylase activity of EglN1, EglN2 and EglN3. We hypothesized that the qualitatively different effects of R- and S-2HG on the EglN prolyl-4-hydroxylases might influence their transforming activities. In order to elucidate the role of mutant IDH, and R- and S-2HG, in myeloid leukemia, we developed a myeloid transformation assay using TF-1 cells. TF-1 is a human erythroleukemia cell line that requires GM-CSF for growth and undergoes erythrocytic differentiation when stimulated with erythropoietin (EPO). We expressed wild-type IDH1 (WTIDH1), a tumor-derived mutant IDH1 (IDH1R132H), or a catalytically inactive IDH1R132H variant (IDH1R132H/3DN) in TF-1 cells. As expected, cells expressing IDH1R132H, but not cells expressing WTIDH1 or IDH1R132H/3DN, had dramatically elevated levels of 2HG. Furthermore, we found that expression of IDH1R132H, but not WTIDH1 or IDH1R132H/3DN, conferred growth factor-independence to TF-1 cells (Figure 1a), and blocked their EPO-induced differentiation (Figure 1b). In order to determine whether transformation of TF-1 cells by IDH1R132H is mediated by 2HG, we treated TF-1 cells with cell-permeable esterified R-2HG or S-2HG. R-2HG recapitulated the growth and differentiation phenotypes of IDH1R132H expression in a dose-dependent manner. In contrast, S-2HG did not induce these phenotypes at any concentration tested. Next, we examined the effect of loss of TET2 on TF-1 cells. We infected TF-1 cells with shRNAs targeting TET1 or TET2 and found that knockdown of TET2, but not TET1, induced growth factor-independence and blocked EPO-induced differentiation similarly to expression of IDH1R132H or treatment with R-2HG. Interestingly, we found that transformation by IDH1R132H and TET2 knockdown were reversed by inhibition of EglN1 (Figure 2), suggesting that R-2HG, but not S-2HG, transforms leukemic cells by inhibiting targets such as TET2 while preserving, and possibly enhancing, EglN activity. These findings further suggest that therapeutic targeting of EglN prolyl-4-hydroxylase activity might be effective in the treatment of IDH1-mutant and TET2-mutant myeloid leukemias. Disclosures: Kaelin: Fibrogen: Consultancy, Equity Ownership.

Enantiomer-Specific Transformation by 2HG Is Linked to Opposing Effects on α-Ketoglutarate-Dependent Dioxygenases

Abstract LBA-4Somatic mutations in IDH1 and IDH2 occur frequently in clonal myeloid disorders and result in the neomorphic ability of IDH to convert α-ketoglutarate (2-OG) to the R-enantiomer of 2-hydroxyglutarate (R-2HG) (Dang, et al Nature 462: 739, 2009). 2OG is an essential cofactor for many metabolic enzymes, including the TET family of 5-methylcytosine hydroxylases and the EglN family of prolyl-4-hydroxylases, and 2HG has been shown to inhibit several 2OG-dependent dioxygenases in vitro, including TET2 (Xu, et al Cancer Cell 19: 17, 2011; Figueroa, et al Cancer Cell 18: 1, 2010). We recently showed that the (S) enantiomer of 2HG (S-2HG), but not the (R) enantiomer of 2HG (R-2HG), inhibits the EglN prolyl-4-hydroxylases (Koivunen, et al. Submitted for publication). Moreover, we found that R-2HG can act as a cofactor to promote the hydroxylase activity of EglN1, EglN2 and EglN3. We hypothesized that the qualitatively different effects of R- and S-2HG on the EglN prolyl-4-hydroxylases might influence their transforming activities.In order to elucidate the role of mutant IDH, and R- and S-2HG, in myeloid leukemia, we developed a myeloid transformation assay using TF-1 cells. TF-1 is a human erythroleukemia cell line that requires GM-CSF for growth and undergoes erythrocytic differentiation when stimulated with erythropoietin (EPO). We expressed wild-type IDH1 (WTIDH1), a tumor-derived mutant IDH1 (IDH1R132H), or a catalytically inactive IDH1R132H variant (IDH1R132H/3DN) in TF-1 cells. As expected, cells expressing IDH1R132H, but not cells expressing WTIDH1 or IDH1R132H/3DN, had dramatically elevated levels of 2HG. Furthermore, we found that expression of IDH1R132H, but not WTIDH1 or IDH1R132H/3DN, conferred growth factor-independence to TF-1 cells (Figure 1a), and blocked their EPO-induced differentiation (Figure 1b). In order to determine whether transformation of TF-1 cells by IDH1R132H is mediated by 2HG, we treated TF-1 cells with cell-permeable esterified R-2HG or S-2HG. R-2HG recapitulated the growth and differentiation phenotypes of IDH1R132H expression in a dose-dependent manner. In contrast, S-2HG did not induce these phenotypes at any concentration tested. Next, we examined the effect of loss of TET2 on TF-1 cells. We infected TF-1 cells with shRNAs targeting TET1 or TET2 and found that knockdown of TET2, but not TET1, induced growth factor-independence and blocked EPO-induced differentiation similarly to expression of IDH1R132H or treatment with R-2HG. Interestingly, we found that transformation by IDH1R132H and TET2 knockdown were reversed by inhibition of EglN1 (Figure 2), suggesting that R-2HG, but not S-2HG, transforms leukemic cells by inhibiting targets such as TET2 while preserving, and possibly enhancing, EglN activity. These findings further suggest that therapeutic targeting of EglN prolyl-4-hydroxylase activity might be effective in the treatment of IDH1-mutant and TET2-mutant myeloid leukemias. [Display omitted] Disclosures:Kaelin:Fibrogen: Consultancy, Equity Ownership.

Design, synthesis, and biological evaluation of a novel series of bisintercalating DNA-binding piperazine-linked bisanthrapyrazole compounds as anticancer agents

A series of bisintercalating DNA binding bisanthrapyrazole compounds containing piperazine linkers were designed by molecular modeling and docking techniques. Because the anthrapyrazoles are not quinones they are unable to be reductively activated like doxorubicin and other anthracyclines and thus they should not be cardiotoxic. The concentration dependent increase in DNA melting temperature was used to determine the strength of DNA binding and the bisintercalation potential of the compounds. Compounds with more than a three-carbon linker that could span four DNA base pairs achieved bisintercalation. All of the bisanthrapyrazoles inhibited human erythroleukemic K562 cell growth in the low to submicromolar concentration range. They also strongly inhibited the decatenation activity of topoisomerase IIα and the relaxation activity of topoisomerase I. However, as measured by their ability to induce double strand breaks in plasmid DNA, the bisanthrapyrazole compounds did not act as topoisomerase IIα poisons. In conclusion, a novel group of bisanthrapyrazole compounds were designed, synthesized, and biologically evaluated as potential anticancer agents.

Ethyl acetate extract and its major constituent, isorhamnetin 3-O-rutinoside, from Nitraria retusa leaves, promote apoptosis of human myelogenous erythroleukaemia cells

Fractionation of ethyl acetate extract (EA) obtained from Nitraria retusa leaves was assessed using different methods of chromatography, and isorhamnetin3-O-rutinoside (I3-O-R) was isolated from this extract. Its structure was determined using data obtained from (1) H and (13) C NMR spectra, as well as by various correlation experiments (COSY, HMQC and HMBC). Both EA extract and I3-O-R were investigated for their ability to induce apoptosis in human chronic myelogenous erythroleukaemia cells (K562). Apoptosis of cells from the K562 line was detected by DNA fragmentation, PARP cleavage and by evaluating activities of caspases 3 and 8. Apoptosis, revealed by DNA fragmentation and PARP cleavage, was observed after 48-h incubation of these human myelogenous erythroleukaemia cells (K562), with the tested products. Likewise, caspase 3 and caspase 8 activities were induced in the presence of the EA extract and I3-O-R after 48 h of incubation. Our results strongly suggest the involvement of the extrinsic pathway of apoptosis in cells treated by both the original EA extract and its major component, I3-O-R.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.

Cell Death Induced by the Jak2 Inhibitor, G6, Correlates with Cleavage of Vimentin Filaments

Hyperkinetic Jak2 tyrosine kinase signaling has been implicated in several human diseases including leukemia, lymphoma, myeloma, and the myeloproliferative neoplasms. Using structure-based virtual screening, we previously identified a novel Jak2 inhibitor named G6. We showed that G6 specifically inhibits Jak2 kinase activity and suppresses Jak2-mediated cellular proliferation. To elucidate the molecular and biochemical mechanisms by which G6 inhibits Jak2-mediated cellular proliferation, we treated Jak2-V617F expressing human erythroleukemia (HEL) cells for 12 h with either vehicle control or 25 μM of the drug and compared protein expression profiles using two-dimensional gel electrophoresis. One differentially expressed protein identified by electrospray mass spectroscopy was the intermediate filament protein, vimentin. It was present in DMSO treated cells but absent in G6 treated cells. HEL cells treated with G6 showed both time- and dose-dependent cleavage of vimentin as well as a marked reorganization of vimentin intermediate filaments within intact cells. In a mouse model of Jak2-V617F mediated human erythroleukemia, G6 also decreased the levels of vimentin protein, in vivo. The G6-induced cleavage of vimentin was found to be Jak2-dependent and calpain-mediated. Furthermore, we found that intracellular calcium mobilization is essential and sufficient for the cleavage of vimentin. Finally, we show that the cleavage of vimentin intermediate filaments, per se, is sufficient to reduce HEL cell viability. Collectively, these results suggest that G6-induced inhibition of Jak2-mediated pathogenic cell growth is concomitant with the disruption of intracellular vimentin filaments. As such, this work describes a novel pathway for the targeting of Jak2-mediated pathological cell growth.

Knockdown of hTERT Alters Biophysical Properties of K562 Cells Resulting in Decreased Migration Rate In Vitro

It has been shown that 90% of tumors, including hematological malignant tumors and leukemia, have much higher levels of telomerase expression than normal cells. To investigate the effect of telomerase on leukemia cells, we transfected K562, a human erythroleukemia cell line with an antisense-hTERT (human telomerase reverse transcriptase) cDNA vector, and examined the biological and biophysical properties of the stably transfected cells (referred to as KAT). Un-transfected cells (K562) and cells transfected with the empty vector (referred to as KC) were used as controls. Cell growth curve and (3)H-TdR test showed that the growth rate and DNA synthesis of KAT decreased compared with those of K562 and KC cells. Apoptosis and cell cycle distribution in KAT cells under normal culture condition were similar to those of K562 and KC cells, but changed after serum deprivation. KAT cells had significantly different biophysical characteristics from K562 and KC in terms of cell electrophoresis, membrane fluidity, membrane fluidity, and viscoelasticity. Furthermore, the transendothelial migration rate of KAT was much lower than those of K562 and KC cells. Confocal microscopy showed that KAT cells had higher F-actin content, suggesting the reorganization of cytoskeleton. Flow cytometry analysis revealed a lowered intracellular calcium concentration and CD71 expression, explaining the high F-actin content in KAT cells. In conclusion, we found that the knockdown of hTERT in K562 cells changed their cytoskeleton and biophysical features, and reduced the cell migration.

α1-adrenergic drugs exhibit affinity to a thapsigargin-sensitive binding site and interfere with the intracellular Ca 2+ homeostasis in human erythroleukemia cells

Even though the erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors, some α1-adrenergic drugs influence both survival and differentiation of these cell lines. Since Ca 2+ is closely related to cellular homeostasis, we examined the capacity of α1-adrenergic drugs to modulate the intracellular Ca 2+ content in K562 cells. Because of morphological alterations of mitochondria following α1-adrenergic agonist treatment, we also scrutinized mitochondrial functions. In order to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells, we evaluated the application of the fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin. We discovered that the α1-adrenergic agonists naphazoline, oxymetazoline and also the α1-adrenergic antagonist benoxathian are able to raise the intracellular Ca 2+-content in K562 cells. Furthermore, we demonstrate that naphazoline treatment induces ROS-formation as well as an increase in Δψm in K562 cells. Using BODIPY® FL-Prazosin we were able to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells. Interestingly, the SERCA-inhibitor thapsigargin appears to interfere with the binding of BODIPY® FL-Prazosin. Our data suggest that the effects of α1-adrenergic drugs on erythroleukemia cells are mediated by a thapsigargin sensitive binding site, which controls the fate of erythroleukemia cells towards differentiation, senescence and cell death through modulation of intracellular Ca 2+.

MiR-34a Regulates Mouse Neural Stem Cell Differentiation

BackgroundMicroRNAs (miRNAs or miRs) participate in the regulation of several biological processes, including cell differentiation. Recently, miR-34a has been implicated in the differentiation of monocyte-derived dendritic cells, human erythroleukemia cells, and mouse embryonic stem cells. In addition, members of the miR-34 family have been identified as direct p53 targets. However, the function of miR-34a in the control of the differentiation program of specific neural cell types remains largely unknown. Here, we investigated the role of miR-34a in regulating mouse neural stem (NS) cell differentiation.Methodology/Principal FindingsmiR-34a overexpression increased postmitotic neurons and neurite elongation of mouse NS cells, whereas anti-miR-34a had the opposite effect. SIRT1 was identified as a target of miR-34a, which may mediate the effect of miR-34a on neurite elongation. In addition, acetylation of p53 (Lys 379) and p53-DNA binding activity were increased and cell death unchanged after miR-34a overexpression, thus reinforcing the role of p53 during neural differentiation. Interestingly, in conditions where SIRT1 was activated by pharmacologic treatment with resveratrol, miR-34a promoted astrocytic differentiation, through a SIRT1-independent mechanism.ConclusionsOur results provide new insight into the molecular mechanisms by which miR-34a modulates neural differentiation, suggesting that miR-34a is required for proper neuronal differentiation, in part, by targeting SIRT1 and modulating p53 activity.

α1-adrenergic drugs modulate differentiation and cell death of human erythroleukemia cells through non adrenergic mechanism

Preliminary data showed that α1-adrenergic antagonists induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells. To test the hypothesis whether survival and differentiation of erythroleukemia cells are under control of α1-adrenergic signalling, we examined α1-adrenoceptor expression of erythroleukemia cells and compared the in vitro effects of α-adrenergic antagonists with those of agonists. We discovered that α1-adrenergic agonists suppress both erythroid differentiation and growth of erythroleukemia cells concomitant with lipofuscin accumulation, autophagy and necrotic cell death. α1-adrenergic agonists also inhibit the in vitro growth of physiologic hematopoietic progenitors obtained from umbilical cord blood with high selectivity for the erythroid lineage. Interestingly, the observed effects could not be related to α1-adrenoceptors, even though agonists and antagonists displayed opposing effects regarding cellular growth and differentiation of erythroleukemia cells. Our data suggest that the effects of α1-adrenergic drugs are related to a non-adrenoceptor binding site, controlling the fate of erythroid progenitor cells towards differentiation and cell death. Since the observed effects are not mediated through adrenoceptors, the physiologic relevance of our data remains unclear, so far. Nevertheless, the identification of the still unknown binding site(s) might disclose new insights into regulation of erythroid differentiation and cell death.

Chemical and Biological Activity of Leaf Extracts of Chromolaena Leivensis

The flavonoids 3,5-dihydroxy-7-methoxy-flavanone, 3,5-dihydroxy-7-methoxyflavone and 3,5,7-trihydroxy-6-methoxyflavone were isolated from the leaves of C. leivensis. Preliminary observations in K562 cells (human erythroleukemia) using the trypan blue test, showed a 90% viability at a concentration of 100 microg/mL; however, further testing of the flavonoids at concentrations of 25, 50 and 100 microg/mL showed toxicity affecting the morphology of human erythroleukemia cells (K562) and human melanoma cells (A375). Induction of apoptosis was produced by 3,5-dihydroxy-7-methoxyflavone at 72 hours after treatment with arrest in the G2 / M phase of the cell cycle. The A375 cells treated with 50 microg/mL of 3,5-dihydroxy-7-methoxy-flavanone for 24, 48 and 72 hours, display effects on the behavior of the cell cycle. The flavonoid 3,5-dihydroxy-7-methoxyflavone has activity on the mitochondrial membrane at concentrations of 25, 50 and 100 microg/mL, at time intervals of 8 to 12 hours. The flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-dihydroxy-7-methoxyflavone at a concentration of 25 microg/mL increased the expression of costimulatory molecules corresponding to the phenotype presented by mature dendritic cells with differentiation markers CD40, CD83, CD86 and HLA-DR. The two flavonoids at concentrations between 0.39 and 100 microg/mL slightly increased the proliferation of peripheral blood mononuclear cells in the presence and in the absence of phytohemagglutinin. These flavonoids at concentrations of 50 and 100 microg/mL slightly increased the proliferation of fibroblasts.