Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

PubMed

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Osthole shows the potential to overcome P-glycoprotein‑mediated multidrug resistance in human myelogenous leukemia K562/ADM cells by inhibiting the PI3K/Akt signaling pathway.

PubMed

Wang, Hong; Jia, Xiu-Hong; Chen, Jie-Ru; Wang, Jian-Yong; Li, You-Jie

2016-06-01

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) has been reported to play a pivotal role in tumor chemotherapy failure. Study after study has illustrated that the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is involved in the MDR phenotype and is correlated with P-gp expression in many human malignancies. In the present study, osthole, an O-methylated coumarin, exhibited potent reversal capability of MDR in myelogenous leukemia K562/ADM cells. Simultaneously, the uptake and efflux of Rhodamine-123 (Rh-123) and the accumulation of doxorubicin assays combined with flow cytometric analysis suggested that osthole could increase intracellular drug accumulation. Furthermore, osthole decreased the expression of multidrug resistance gene 1 (MDR1) at both the mRNA and protein levels. Further experiments elucidated that osthole could suppress P-gp expression by inhibiting the PI3K/Akt signaling pathway which might be the main mechanism accounting for the reversal potential of osthole in the MDR in K562/ADM cells. In conclusion, osthole combats MDR and could be a promising candidate for the development of novel MDR reversal modulators.

Involvement of p38 MAPK- and JNK-modulated expression of Bcl-2 and Bax in Naja nigricollis CMS-9-induced apoptosis of human leukemia K562 cells.

PubMed

Chen, Ying-Jung; Liu, Wen-Hsin; Kao, Pei-Hsiu; Wang, Jeh-Jeng; Chang, Long-Sen

2010-06-15

CMS-9, a phospholipase A(2) (PLA(2)) isolated from Naja nigricollis venom, induced apoptosis of human leukemia K562 cells, characterized by mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Moreover, an increase in intracellular Ca2+ concentration and the production of reactive oxygen species (ROS) was noted. Pretreatment with BAPTA-AM (Ca2+ chelator) and N-acetylcysteine (NAC, ROS scavenger) proved that Ca2+ was an upstream event in inducing ROS generation. Upon exposure to CMS-9, activation of p38 MAPK and JNK was observed in K562 cells. BAPTA-AM or NAC abrogated CMS-9-elicited p38 MAPK and JNK activation, and rescued viability of CMS-9-treated K562 cells. SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor) suppressed CMS-9-induced dissipation of mitochondrial membrane potential, Bcl-2 down-regulation, Bax up-regulation and increased mitochondrial translocation of Bax. Inactivation of PLA(2) activity reduced drastically the cytotoxicity of CMS-9, and a combination of lysophosphatidylcholine and stearic acid mimicked the cytotoxic effects of CMS-9. Taken together, our data suggest that CMS-9-induced apoptosis of K562 cells is catalytic activity-dependent and is mediated through mitochondria-mediated death pathway triggered by Ca2+/ROS-evoked p38 MAPK and JNK activation. 2010 Elsevier Ltd. All rights reserved.

Phenethyl isothiocyanate inhibits growth of human chronic myeloid leukemia K562 cells via reactive oxygen species generation and caspases.

PubMed

Wang, Yating; Wei, Sixi; Wang, Jishi; Fang, Qin; Chai, Qixiang

2014-07-01

Phenethyl isothiocyanate (PEITC), a potential cancer chemopreventive constituent of cruciferous vegetables, including watercress, has been reported to inhibit cancer cell growth by arresting the cell cycle and inducing apoptosis in various human cancer cell models. However, the role of PEITC in the inhibition of human chronic myeloid leukemia (CML) K562 cell growth and its underlying mechanisms have yet to be elucidated. In the present study, PEITC was found to induce cell death through the induction of reactive oxygen species (ROS) stress and oxidative damage. Heme oxygenase‑1 (HO‑1), which participates in the development of numerous tumors and the sensitivity of these tumors to chemotherapeutic drugs, plays a protective role by modulating oxidative injury. Therefore, the present study assessed the inhibitory effect of PEITC on K562 cells and whether HO‑1 facilitated cell apoptosis and ROS generation. PEITC was found to suppress cell growth and cause apoptosis by promoting Fas and Fas ligand expression, increasing ROS generation and by the successive release of cytochrome c as well as the activation of caspase‑9 and caspase‑3. PEITC was also combined with the HO‑1 inhibitor zinc protoporphyrin IX and the inducer hemin to assess whether HO‑1 determines cell survival and ROS generation. The results of the present study suggest that PEITC may be a potential anti‑tumor compound for CML therapy, and that HO‑1 has a critical function in PEITC‑induced apoptosis and ROS generation.

Oxidative stress by ascorbate/menadione association kills K562 human chronic myelogenous leukaemia cells and inhibits its tumour growth in nude mice.

PubMed

Verrax, Julien; Stockis, Julie; Tison, Aurélie; Taper, Henryk S; Calderon, Pedro Buc

2006-09-14

The effect of oxidative stress induced by the ascorbate/menadione-redox association was examined in K562 cells, a human erythromyeloid leukaemia cell line. Our results show that ascorbate enhances menadione redox cycling, leading to the formation of intracellular reactive oxygen species (as shown by dihydrorhodamine 123 oxidation). The incubation of cells in the presence of both ascorbate/menadione and aminotriazole, a catalase inhibitor, resulted in a strong decrease of cell survival, reinforcing the role of H(2)O(2) as the main oxidizing agent killing K562 cells. This cell death was not caspase-3-dependent. Indeed, neither procaspase-3 and PARP were processed and only a weak cytochrome c release was observed. Moreover, we observed only 23% of cells with depolarized mitochondria. In ascorbate/menadione-treated cells, DNA fragmentation was observed without any sign of chromatin condensation (DAPI and TUNEL tests). The cell demise by ascorbate/menadione is consistent with a necrosis-like cell death confirmed by both cytometric profile of annexin-V/propidium iodide labeled cells and by light microscopy examination. Finally, we showed that a single i.p. administration of the association of ascorbate and menadione is able to inhibit the growth of K562 cells by about 60% (in both tumour size and volume) in an immune-deficient mice model. Taken together, these results reinforced our previous claims about a potential application of the ascorbate/menadione association in cancer therapy.

Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

PubMed Central

Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

2015-01-01

Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

Synthesis of a Novel Series of 2-Methylsulfanyl Fatty Acids and their Toxicity on the Human K-562 and U-937 Leukemia Cell Lines

PubMed Central

Carballeira, Néstor M.; Miranda, Carlos; Orellano, Elsie A.; González, Fernando A.

2006-01-01

The hitherto unknown 2-methylsulfanyldecanoic acid and 2-methylsulfanyldodecanoic acid were synthesized from methyl decanoate and methyl dodecanoate, respectively, through the reaction of lithium diisopropylamide and dimethyldisulfide in THF followed by saponification with potassium hydroxide in ethanol. Both α-methylsulfanylated FA were cytotoxic to the human chronic myelogenous leukemia K-562 and the human histiocytic lymphoma U-937 cell lines with EC50 values in the 200-300 μM range, which makes them more cytotoxic to these cell lines than either decanoic acid or dodecanoic acid. The cytotoxicity of the studied FA towards K-562 followed the order: 2-SCH3-12:0 > 2-SCH3-10:0 > 10:0 > 12:0 > 2-OCH3-12:0, while towards U-937 the cytotoxicity was found to be: 2-SCH3-10:0 > 2-SCH3-12:0 > 12:0 > 10:0 > 2-OCH3-12:0. These results indicate that the α-methylsulfanyl substitution increases the cytotoxicity of the C10 and C12 fatty acids towards the studied leukemia cell lines. PMID:16382579

The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming

2012-11-15

Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catecholmore » enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.« less

[Effects of Aptamer-siRNA Nucleic Acid Compound on Growth and Apoptosis in Myeloid Leukemia Cell Line K562].

PubMed

Ping, Juan; Shen, Zhi-Hui; Wang, Bao-Quan; Zhao, Na; Li, Rui; Li, Mian; Pang, Xiao-Bin; Chen, Chuan-Bo

2015-04-01

To explore the effects of aptamer-siRNA nucleic acid compound on growth and apoptosis in myeloid leukemia cell line K562. the changes of cellular morphology and structure were observed by using fluorescence microscope, laser confocal microscope, JEM-4000EX transmission electron microscopy; MTT assay were performed to evaluate the sensibility of K562 cells to aptamer-siRNA compound, the apoptosis was detected by DNA gel electro-phoresis. The remarkably changes of morphology and structure of K562 cells treated with 200 µmol/L aptamer-siRNA were observed under fluorescence microscopy and electromicroscopy. As compared with control, the aptamer-siRNA compound showed more inhibitory effect on K562 cells and there was significant difference (P<0.05). The MTT assay showed that the IC50 value of aptamer-siRNA compound for K562 cells was 150 µmol/L. According to agarose gel electrophoresis observation, when the aptamer-siRNA compound showed effect on K562 cells, the typical DNA lader could be observed. The aptamer-siRNA compound can significantly induce K562 cell apoptosis, and provide reference for gene therapy of patients with chronic myelocytic lenkemia.

Diverse Effects of Glutathione and UPF Peptides on Antioxidant Defense System in Human Erythroleukemia Cells K562.

PubMed

Kairane, Ceslava; Mahlapuu, Riina; Ehrlich, Kersti; Kilk, Kalle; Zilmer, Mihkel; Soomets, Ursel

2012-01-01

The main goal of the present paper was to examine the influence of the replacement of γ-Glu moiety to α-Glu in glutathione and in its antioxidative tetrapeptidic analogue UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), resulting in α-GSH and UPF17 (Tyr(Me)-Glu-Cys-Gly), on the antioxidative defense system in K562 cells. UPF1 and GSH increased while UPF17 and α-GSH decreased the activity of CuZnSOD in K562 cells, at peptide concentration of 10 μM by 42% and 38% or 35% and 24%, respectively. After three-hour incubation, UPF1 increased and UPF17 decreased the intracellular level of total GSH. Additionally, it was shown that UPF1 is not degraded by γ-glutamyltranspeptidase, which performs glutathione breakdown. These results indicate that effective antioxidative character of peptides does not depend only on the reactivity of the thiol group, but also of the other functional groups, and on the spatial structure of peptides.

Electrochemical K-562 cells sensor based on origami paper device for point-of-care testing.

PubMed

Ge, Shenguang; Zhang, Lina; Zhang, Yan; Liu, Haiyun; Huang, Jiadong; Yan, Mei; Yu, Jinghua

2015-12-01

A low-cost, simple, portable and sensitive paper-based electrochemical sensor was established for the detection of K-562 cell in point-of-care testing. The hybrid material of 3D Au nanoparticles/graphene (3D Au NPs/GN) with high specific surface area and ionic liquid (IL) with widened electrochemical windows improved the good biocompatibility and high conductivity was modified on paper working electrode (PWE) by the classic assembly method and then employed as the sensing surface. IL could not only enhance the electron transfer ability but also provide sensing recognition interface for the conjugation of Con A with cells, with the cell capture efficiency and the sensitivity of biosensor strengthened simultaneously. Concanavalin A (Con A) immobilization matrix was used to capture cells. As proof-of-concept, the paper-based electrochemical sensor for the detection of K-562 cells was developed. With such sandwich-type assay format, K-562 cells as model cells were captured on the surface of Con A/IL/3D AuNPs@GN/PWE. Con A-labeled dendritic PdAg NPs were captured on the surface of K-562 cells. Such dendritic PdAg NPs worked as catalysts promoting the oxidation of thionine (TH) by H2O2 which was released from K-562 cells via the stimulation of phorbol 12-myristate-13-acetate (PMA). Therefore, the current signal response was dependent on the amount of PdAg NPs and the concentration of H2O2, the latter of which corresponded with the releasing amount from cells. So, the detection method of K-562 cell was also developed. Under optimized experimental conditions, 1.5×10(-14) mol of H2O2 releasing from each cell was calculated. The linear range and the detection limit for K-562 cells were determined to be 1.0×10(3)-5.0×10(6) cells/mL and 200 cells/mL, respectively. Such as-prepared sensor showed excellent analytical performance with good fabrication reproducibility, acceptable precision and satisfied accuracy, providing a novel protocol in point-of-care testing of cells

Olive (Olea europaea) Leaf Extract Induces Apoptosis and Monocyte/Macrophage Differentiation in Human Chronic Myelogenous Leukemia K562 Cells: Insight into the Underlying Mechanism

PubMed Central

Han, Junkyu; Jlaiel, Lobna; Sayadi, Sami; Isoda, Hiroko

2014-01-01

Differentiation therapy is an attractive approach aiming at reversing malignancy and reactivating endogenous differentiation programs in cancer cells. Olive leaf extract, known for its antioxidant activity, has been demonstrated to induce apoptosis in several cancer cells. However, its differentiation inducing properties and the mechanisms involved are still poorly understood. In this study, we investigated the effect of Chemlali Olive Leaf Extract (COLE) for its potential differentiation inducing effect on multipotent leukemia K562 cells. Results showed that COLE inhibits K562 cells proliferation and arrests the cell cycle at G0/G1, and then at G2/M phase over treatment time. Further analysis revealed that COLE induces apoptosis and differentiation of K562 cells toward the monocyte lineage. Microarray analysis was conducted to investigate the underlying mechanism of COLE differentiation inducing effect. The differentially expressed genes such as IFI16, EGR1, NFYA, FOXP1, CXCL2, CXCL3, and CXCL8 confirmed the commitment of K562 cells to the monocyte/macrophage lineage. Thus our results provide evidence that, in addition to apoptosis, induction of differentiation is one of the possible therapeutic effects of olive leaf in cancer cells. PMID:24803988

Olive (Olea europaea) leaf extract induces apoptosis and monocyte/macrophage differentiation in human chronic myelogenous leukemia K562 cells: insight into the underlying mechanism.

PubMed

Samet, Imen; Han, Junkyu; Jlaiel, Lobna; Sayadi, Sami; Isoda, Hiroko

2014-01-01

Differentiation therapy is an attractive approach aiming at reversing malignancy and reactivating endogenous differentiation programs in cancer cells. Olive leaf extract, known for its antioxidant activity, has been demonstrated to induce apoptosis in several cancer cells. However, its differentiation inducing properties and the mechanisms involved are still poorly understood. In this study, we investigated the effect of Chemlali Olive Leaf Extract (COLE) for its potential differentiation inducing effect on multipotent leukemia K562 cells. Results showed that COLE inhibits K562 cells proliferation and arrests the cell cycle at G0/G1, and then at G2/M phase over treatment time. Further analysis revealed that COLE induces apoptosis and differentiation of K562 cells toward the monocyte lineage. Microarray analysis was conducted to investigate the underlying mechanism of COLE differentiation inducing effect. The differentially expressed genes such as IFI16, EGR1, NFYA, FOXP1, CXCL2, CXCL3, and CXCL8 confirmed the commitment of K562 cells to the monocyte/macrophage lineage. Thus our results provide evidence that, in addition to apoptosis, induction of differentiation is one of the possible therapeutic effects of olive leaf in cancer cells.

Acute erythremic myelosis (true erythroleukaemia): a variant of AML FAB-M6.

PubMed

Hasserjian, R P; Howard, J; Wood, A; Henry, K; Bain, B

2001-03-01

Classic erythroleukaemia (acute myeloid leukaemia M6, or M6 AML) is defined as an excess of myeloblasts in an erythroid predominant background. Leukaemia variants in which the primitive blast cells are demonstrably erythroid are extremely rare and poorly characterised. Variably referred to as "true erythroleukaemia" or "acute erythremic myelosis", they are often included within the M6 AML category even though they do not meet strict criteria for this type of AML. Two cases of acute erythroid neoplasia are presented with clinical, morphological, immunophenotypic, and cytogenetic analysis. Both patients presented with profound anaemia, one in a setting of long standing myelodysplasia. Bone marrow examination revealed a predominant population of highly dysplastic erythroid cells in both cases. In one case, the liver was infiltrated by neoplastic erythroid cells. Both patients died within four months of diagnosis. This report illustrates that cases of acute leukaemia occur in which the dominant neoplastic cell is a primitive erythroid cell without an accompanying increase in myeloblasts. This does not preclude the neoplastic clone originating in a multipotent haemopoietic stem cell, as suggested by cases arising in patients with myelodysplasia. Acute erythremic myelosis should be recognised as a distinct variant of M6 AML.

Voltage-Gated K+ Channel, Kv3.3 Is Involved in Hemin-Induced K562 Differentiation

PubMed Central

Song, Min Seok; Choi, Seon Young; Ryu, Pan Dong; Lee, So Yeong

2016-01-01

Voltage-gated K+ (Kv) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between Kv channels and cell differentiation remains poorly investigated. This study demonstrates that Kv3.3 is involved in K562 cell erythroid differentiation. Down-regulation of Kv3.3 using siRNA-Kv3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of Kv3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The Kv channels, or at least Kv3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of Kv channel regulation of cell differentiation would provide important information regarding vital cellular processes. PMID:26849432

Research on the effect of formononetin on photodynamic therapy in K562 cells.

PubMed

Sun, Dan; Lu, Yao; Zhang, Su-Juan; Wang, Kai-Ge; Sun, Zhe

2017-10-01

At the present time, many cancer patients combine some forms of complementary and alternative medicine therapies with their conventional therapies. The most common choice of these therapies is the use of antioxidants. Formononetin is presented in different foods. It has a variety of biological activities including antioxidant and anti-cancer properties. On account of its antioxidant activity, formononetin might protect cancer cells from free radical damage in photodynamic therapy (PDT) during which reactive oxygen species (ROS) production was stimulated leading to irreversible tumor cell injury. In this study, the influence of formononetin on K562 cells in PDT was demonstrated. The results showed that formononetin supplementation alone did not affect the lipid peroxidation, DNA damage and apoptosis in K562 cells. It increases the lipid peroxidation, DNA damage and apoptosis in K562 cells induced by PDT. The singlet oxygen quencher sodium azide suppresses the apoptosis induced by PDT with formononetin. In conclusion, formononetin consumption during PDT increases the effectiveness of cancer therapy on malignant cells. The effect of antioxidants on PDT maybe was determined by its sensitization ability to singlet oxygen.

Overexpression of Hiwi Inhibits the Cell Growth of Chronic Myeloid Leukemia K562 Cells and Enhances Their Chemosensitivity to Daunomycin.

PubMed

Wang, Yalin; Jiang, Yan; Bian, Cuicui; Dong, Yi; Ma, Chao; Hu, Xiaolin; Liu, Ziling

2015-09-01

Chronic myeloid leukemia (CML) is a clonal disorder characterized by excessive accumulation of myeloid cells in the peripheral blood. In the present study, to investigate the role of Hiwi in leukemogenesis, lentivirus-mediated Hiwi overexpression was performed in a CML cell line, K562 cells. Our data revealed that Hiwi protein expression was undetectable in K562 cells, and its overexpression suppressed cell proliferation, induced cell cycle arrest at G0/G1 and G2/M phases, and promoted apoptosis in K562 cells in vitro. Expression of anti-apoptotic protein, Bcl-2, was decreased in cells expressing Hiwi, whereas that of pro-apoptotic proteins, Bax, activated caspase-3, -9, and cleaved poly (ADP-ribose) polymerase were increased. Additionally, Hiwi upregulation enhanced the chemosensitivity of CML cells to daunomycin. Our study illustrates that expression deletion of Hiwi may be involved in the pathogenesis of human CML and suggests a possible role of Hiwi in regulating the cell growth, cell cycle, and apoptosis of CML cells in vitro.

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed Central

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-01-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-03-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

Expression of transcription factors during sodium phenylacetate induced erythroid differentiation in K562 cells.

PubMed

Rath, A V; Schmahl, G E; Niemeyer, C M

1997-01-01

During 15 days of treatment of K562 cells with sodium phenylacetate, we observed an increase in the cellular hemoglobin concentration with a similar increase in the expression of gamma-globin mRNA. Morphological studies demonstrated characteristic features of erythroid differentiation and maturation. At the same time there was no change in the level of expression of the cell surface antigenes CD33, CD34, CD45, CD71 and glycophorin A. Likewise, the level of expression of the erythroid transcription factors GATA-1, GATA-2, NF-E2, SCL and RBTN2, all expressed in untreated K562 cells, did not increase during sodium phenylacetate induced erythroid differentiation. The expression of the nuclear factors Evi-1 and c-myb, known to inhibit erythroid differentiation, did not decrease. We conclude that sodium phenylacetate treatment of K562 cells increases gamma-globin mRNA and induces cell maturation as judged by morphology without affecting the expression of the erythroid transcription factors, some of which are known to be involved in the regulation of beta-like globin genes.

Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA

PubMed Central

Ojima, Yoshihiro; Duncan, Mark Thompson; Nurhayati, Retno Wahyu; Taya, Masahito; Miller, William Martin

2013-01-01

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation. PMID:23770036

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

PubMed

Valletta, Elena; Rinaldi, Annamaria; Marini, Mario; Franzese, Ornella; Roscetti, Gianna

2018-05-22

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 48 hr 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. Copyright © 2018 John Wiley & Sons, Ltd.

Reversal effect of a macrocyclic bisbibenzyl plagiochin E on multidrug resistance in adriamycin-resistant K562/A02 cells.

PubMed

Shi, Yan-Qiu; Qu, Xian-Jun; Liao, Yong-Xiang; Xie, Chun-Feng; Cheng, Yan-Na; Li, Song; Lou, Hong-Xiang

2008-04-14

Plagiochin E is a new macrocyclic bisbibenzyl compound isolated from Marchantia polymorpha. In the previous studies, we reported that when combined with fluconazole, plagiochin E had synergetic effects against the resistant strain of Candida albicans. Herein, we examined the reversal effect of plagiochin E on multidrug resistance in adriamycin-induced resistant K562/A02 cells and the parental K562 cells. Its cytotoxicity and reversal effects on multidrug resistance were assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) assay. Apoptosis percentage of cells was obtained from Annexin V/fluorescein isothiocyanate (FITC) and propridium iodide (PI) double-staining. The effects of plagiochin E on P-glycoprotein activity were evaluated by measuring rhodamine 123 (Rh123)-associated mean fluorescence intensity and P-glycoprotein expression on the basis of the flow cytometric technology, respectively. The results showed that plagiochin E ranging from 2 to 12 mug/ml had little cytotoxicity against K562/A02 cells. When combined with adriamycin, it significantly promoted the sensitivity of K562/A02 cells toward adriamycin through increasing intracellular accumulation of adriamycin in a dose-dependent manner. Further study demonstrated that the inhibitory effect of plagiochin E on P-glycoprotein activity was the major cause of increased stagnation of adriamycin inside K562/A02 cells, indicating that plagiochin E, as a new class of mutidrug resistance inhibitor, may effectively reverse the multidrug resistance in K562/A02 cells via inhibiting expression and drug-transport function of P-glycoprotein.

Aclacinomycin A Sensitizes K562 Chronic Myeloid Leukemia Cells to Imatinib through p38MAPK-Mediated Erythroid Differentiation

PubMed Central

Liu, Fu-Hwa; Huang, Yu-Wen; Huang, Huei-Mei

2013-01-01

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and

Aclacinomycin A sensitizes K562 chronic myeloid leukemia cells to imatinib through p38MAPK-mediated erythroid differentiation.

PubMed

Lee, Yueh-Lun; Chen, Chih-Wei; Liu, Fu-Hwa; Huang, Yu-Wen; Huang, Huei-Mei

2013-01-01

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and

Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA.

PubMed

Ojima, Yoshihiro; Duncan, Mark Thompson; Nurhayati, Retno Wahyu; Taya, Masahito; Miller, William Martin

2013-08-15

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation. Copyright © 2013 Elsevier Inc. All rights reserved.

[Effect of Recombinant Adenovirus AdE-SH2-Caspase 8 on the Apoptosis of Imatinib-resistant K562/G01 Cell Line].

PubMed

Wang, Lin; Fei, Chang; Huang, Zheng-Lan; Li, Hui; Liu, Zhang-Lin; Feng, Wen-Li

2015-08-01

To investigate the effect of SH2-Caspase 8 fusion protein expressed by recombinant adenovirus AdE-SH2-Caspase8-HA-GFP (SC) on the apoptosis of K562/G01 cell line, which is a BCR/ABL positive chronic myeloid leukemia cell line and resistant to imatinib. The K562/G01 cell line was infected with AdE-SH2-Caspase 8-HA-GFP adenovirus (SC), then the cells were divided into 3 groups: AdE-SH2m-Caspase 8-HA-GFP (SmC) group, AdE-GFP (CMV) group and PBS group as control. The infection efficiency was observed under fluorescent microscopy and by flow cytometry. The expression of fusion protein SH2-Caspase 8-HA was measured by Western blot. The morphology of the cells detected by Wright's staining. The apoptosis of the cells were detected by flow cytometry and DNA ladder. The expression of Caspase 3 and PARP were detected by Western blot. The infection efficiency of SC on K562/G01 cells was high which was confirmed by fluorescent microscopy and FCM. SH2-Caspase 8-HA fusion protein were expressed correctly in K562/G01 cells. After treatment with SC the apoptosis of K562/G01 cells could be observed by microscopy. The result of FCM showed that early apoptosis of K562/G01 cells increased significantly as compared with control groups (P < 0.05). DNA ladder showed that the classic DNA ladders appeared in K562/G01 cells after treatment with SC. The wester blot detection showed that the expression level of apoptosis-related protein Caspase 3 and PARP increased. The recombinant adenovirus SC expressing SH2-Caspase 8 fusion protein can induces the apoptosis of K562/G01 cells.

Genetically re-engineered K562 cells significantly expand and functionally activate cord blood natural killer cells: Potential for adoptive cellular immunotherapy.

PubMed

Ayello, Janet; Hochberg, Jessica; Flower, Allyson; Chu, Yaya; Baxi, Laxmi V; Quish, William; van de Ven, Carmella; Cairo, Mitchell S

2017-02-01

Natural killer (NK) cells play a significant role in reducing relapse in patients with hematological malignancies after allogeneic stem cell transplantation, but NK cell number and naturally occurring inhibitory signals limit their capability. Interleukin-15 (IL-15) and 4-1BBL are important modulators of NK expansion and functional activation. To overcome these limitations, cord blood mononuclear cells (CB MNCs) were ex vivo expanded for 7 days with genetically modified K562-mbIL15-41BBL (MODK562) or wild-type K562 (WTK562). NK cell expansion; expression of lysosome-associated membrane protein-1 (LAMP-1), granzyme B, and perforin; and in vitro and in vivo cytotoxicity against B-cell non-Hodgkin lymphoma (B-NHL) were evaluated. In vivo tumor growth in B-NHL-xenografted nonobese diabetic severe combined immune deficient (NOD-scid) gamma (NSG) mice was monitored by tumor volume, cell number, and survival. CB MNCs cultured with MODK562 compared with WTK562 demonstrated significantly increased NK expansion (thirty-fivefold, p < 0.05); LAMP-1 (p < 0.05), granzyme B, and perforin expression (p < 0.001); and in vitro cytotoxicity against B-NHL (p < 0.01). Xenografted mice treated with MODK562 CB experienced significantly decreased B-NHL tumor volume (p = 0.0086) and B-NHL cell numbers (p < 0.01) at 5 weeks and significantly increased survival (p < 0.001) at 10 weeks compared with WTK562. In summary, MODK562 significantly enhanced CB NK expansion and cytotoxicity, enhanced survival in a human Burkitt's lymphoma xenograft NSG model, and could be used in the future as adoptive cellular immunotherapy after umbilical CB transplantation. Future directions include expanding anti-CD20 chimeric receptor-modified CB NK cells to enhance B-NHL targeting in vitro and in vivo. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

PaDef defensin from avocado (Persea americana var. drymifolia) is cytotoxic to K562 chronic myeloid leukemia cells through extrinsic apoptosis.

PubMed

Flores-Alvarez, Luis José; Guzmán-Rodríguez, Jaquelina Julia; López-Gómez, Rodolfo; Salgado-Garciglia, Rafael; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2018-06-01

Plant defensins, a group of antimicrobial peptides, show selective cytotoxicity toward cancer cells. However, their mechanisms of action remain poorly understood. Here, we evaluated the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on K562 chronic myeloid leukemia cells and analyzed the pathway involved in the induction of cell death. The defensin PaDef was not cytotoxic against human PBMCs; however, it was cytotoxic for K562 cell line (IC 50  = 97.3 μg/ml) activating apoptosis at 12 h. PaDef did not affect the mitochondrial membrane potential (ΔΨm), neither the transmembranal potential or the release of intracellular calcium. Also, PaDef induced gene expression of caspase 8 (∼2 fold), TNF-α (∼4 fold) and TNFR1 (∼10 fold). In addition, the activation of caspase 8 was detected at 24 h, whereas caspase 9 activity was not modified, suggesting that the extrinsic apoptosis pathway could be activated. In conclusion, PaDef induces apoptosis on K562 cells, which is related to the activation of caspase 8 and involves the participation of TNF-α, which is a novel property for a plant defensin. Copyright © 2018 Elsevier Ltd. All rights reserved.

Apoptosis induction in MV4-11 and K562 human leukemic cells by Pereskia sacharosa (Cactaceae) leaf crude extract.

PubMed

Asmaa, Mat Jusoh Siti; Al-Jamal, Hamid Ali Nagi; Ang, Cheng Yong; Asan, Jamaruddin Mat; Seeni, Azman; Johan, Muhammad Farid

2014-01-01

Pereskia sacharosa is a genus of cacti widely used in folk medicine for cancer-related treatment. Anti-proliferative effects have been studied in recent years against colon, breast, cervical and lung cancer cell lines, with promising results. We here extended study of anti-proliferative effects to a blood malignancy, leukemia. Two leukemic cell lines, MV4-11 (acute myeloid leukemia) and K562 (chronic myeloid leukemia), were studied. IC50 concentrations were determined and apoptosis and cell cycle regulation were studied by flow cytometric analysis. The expression of apoptosis and cell-cycle related regulatory proteins was assessed by Western blotting. P sacharosa inhibited growth of MV4-11 and K562 cells in a dose-dependent manner. The mode of cell death was via induction of intrinsic apoptotic pathways and cell cycle arrest. There was profound up-regulation of cytochrome c, caspases, p21 and p53 expression and repression of Akt and Bcl-2 expression in treated cells. These results suggest that P sacharosa induces leukemic cell death via apoptosis induction and changes in cell cycle checkpoint, thus deserves further study for anti-leukemic potential.

Cytotoxic and apoptotic effects of different extracts of Artemisia biennis Willd. on K562 and HL-60 cell lines

PubMed Central

Tayarani-Najaran, Zahra; Makki, Farideh-Sadat; Alamolhodaei, Nafiseh-Sadat; Mojarrab, Mahdi; Emami, Seyed Ahmad

2017-01-01

Objective(s): Artemisia is a genus of herbs and small shrubs forms an important part of natural vegetation in Iran. It has been reported that several Artemisia species possess anti-proliferative effects. Considering the value of this genus in anti-cancer researches we have chosen Artemisia biennis for cytotoxic and mechanistic studies. Materials and Methods: In this study we have investigated the cytotoxic and apoptotic effects of petroleum ether, dichloromethane, ethyl acetate, ethanol, and ethanol: water (1:1 v/v) extracts of A. biennis Willd. on two cancer human cell lines (K562 and HL-60) and J774 as normal cells. Results: CH2Cl2 extract was found to have the highest anti-proliferative effect on cancer cells. IC50 values obtained in AlamarBlue® assay for CH2Cl2 extract were 64.86 and 54.31 µg/ml on K562 and HL-60 cells respectively. In flow cytometry histogram of the cells treated with CH2Cl2 extract, sub-G1 peak was induced. DNA fragmentation, increased in the level of Bax and cleavage of PARP protein all showed the induction of apoptosis with CH2Cl2 extract after 48 hr contact with cells. Conclusion: The results can corroborate the cytotoxic and apoptotic effects of the CH2Cl2 extract of A. biennis on the K562 and HL-60 cancer cell lines. PMID:28293393

ChIP-seq and ChIP-exo profiling of Pol II, H2A.Z, and H3K4me3 in human K562 cells.

PubMed

Mchaourab, Zenab F; Perreault, Andrea A; Venters, Bryan J

2018-03-06

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.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

PubMed

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-11-07

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

PubMed Central

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-01-01

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein. PMID:27827994

Undifferentiated HL-60 cells internalize an antitumor alkyl ether phospholipid more rapidly than resistant K562 cells.

PubMed

Tsutsumi, T; Tokumura, A; Kitazawa, S

1998-02-05

In this study, we confirmed a previous finding that 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (methyl-PAF) expresses higher antineoplastic activity against the promyelocytic leukemia cell line HL-60, than against the erythroleukemic cell line K562, and intended to clarify the reason for this. Using an albumin back-exchange method, we measured the rates of binding and internalization of [3H]methyl-PAF by HL-60 and K562 cells. We found that methyl-PAF associated very rapidly and to similar extents with the two types of cells at low concentrations of extracellular bovine serum albumin, but that when bound to the cell surface, it was internalized into HL-60 cells faster than into K562 cells. The internalization of methyl-PAF by HL-60 cells was concentration-independent, intracellular ATP-independent and susceptible to thiol group-modifying reagents and cytochalasin B. Thus the inward transbilayer movement of methyl-PAF seems to occur by cytochalasin B-sensitive protein-mediated mechanism based on passive diffusion not requiring energy, in which SH-groups of protein play a critical role. We also found that the internalization of 1-hexadecanoyl-2-(4,4-difluoro-5,7- dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (Bodipy-C5-PC), whose structure resembles that of methyl-PAF, into HL-60 cells was faster than that into K562 cells. Using a combination of an albumin back-exchange method and observation by confocal laser scanning microscopy, we next examined the intracellular distribution of this fluorescent phospholipid probe after its internalization. Intracellular membranes, especially those peripheral to nuclei, were fluorescence-labeled in both HL-60 and K562 cells, but fluorescence of the nuclear membranes was weak, suggesting that this probe seems mainly to accumulate in intracellular granules, and may interact directly with several key enzymes for phospholipid metabolism, leading to cell injury. Because the difference between

Rotenone isolated from Pachyrhizus erosus displays cytotoxicity and genotoxicity in K562 cells.

PubMed

Estrella-Parra, Edgar A; Gomez-Verjan, Juan C; González-Sánchez, Ignacio; Vázquez-Martínez, Edgar Ricardo; Vergara-Castañeda, Edgar; Cerbón, Marco A; Alavez-Solano, Dagoberto; Reyes-Chilpa, Ricardo

2014-01-01

Pachyrhizus erosus (Fabaceae) is a herb commonly known as 'yam bean', which has been cultivated in México since pre-Columbian times for its edible tubers. The seeds are also known for their acaricidal and insecticidal properties due to rotenone and other isoflavonoid contents. Rotenone has exhibited cytotoxic activity against several human tumour cell lines; however, its mechanism of action is still not fully understood. In this study, we determined the cytotoxicity of rotenone isolated from P. erosus seeds on K562 human leukaemia cells. Rotenone exhibited significant cytotoxic activity (IC50 = 13.05 μM), as determined by the MTT assay. Three other isolated isoflavonoids were not cytotoxic. Rotenone genotoxicity was detected using the comet assay. Rotenone induced cell death, and caspase-3 activation as indicated by TUNEL assay, and immunocytofluorescence. Plasmid nicking assay indicated that rotenone does not interact directly with DNA.

c-myb stimulates cell growth by regulation of insulin-like growth factor (IGF) and IGF-binding protein-3 in K562 leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Min-Sun; Kim, Sun-Young; Arunachalam, Sankarganesh

2009-07-17

c-myb plays an important role in the regulation of cell growth and differentiation, and is highly expressed in immature hematopoietic cells. The human chronic myelogenous leukemia cell K562, highly expresses IGF-I, IGF-II, IGF-IR, and IGF-induced cellular proliferation is mediated by IGF-IR. To characterize the impact of c-myb on the IGF-IGFBP-3 axis in leukemia cells, we overexpressed c-myb using an adenovirus gene transfer system in K562 cells. The overexpression of c-myb induced cell proliferation, compared to control, and c-myb induced cell growth was inhibited by anti-IGF-IR antibodies. c-myb overexpression resulted in a significant increase in the expression of IGF-I, IGF-II, andmore » IGF-IR, and a decrease in IGFBP-3 expression. By contrast, disruption of c-myb function by DN-myb overexpression resulted in significant reduction of IGF-I, IGF-II, IGF-IR, and elevation of IGFBP-3 expression. In addition, exogenous IGFBP-3 inhibited the proliferation of K562 cells, and c-myb induced cell growth was blocked by IGFBP-3 overexpression in a dose-dependent manner. The growth-promoting effects of c-myb were mediated through two major intracellular signaling pathways, Akt and Erk. Activation of Akt and Erk by c-myb was completely blocked by IGF-IR and IGFBP-3 antibodies. These findings suggest that c-myb stimulates cell growth, in part, by regulating expression of the components of IGF-IGFBP axis in K562 cells. In addition, disruption of c-myb function by DN-myb may provide a useful strategy for treatment of leukemia.« less

Glycometabolic adaptation mediates the insensitivity of drug-resistant K562/ADM leukaemia cells to adriamycin via the AKT-mTOR/c-Myc signalling pathway.

PubMed

Zhang, Xueyan; Ai, Ziying; Chen, Jing; Yi, Juan; Liu, Zhuan; Zhao, Huaishun; Wei, Hulai

2017-04-01

In human leukaemia, resistance to chemotherapy leads to treatment ineffectiveness or failure. Previous studies have indicated that cancers with increased levels of aerobic glycolysis are insensitive to numerous forms of chemotherapy and respond poorly to radiotherapy. Whether glycolysis serves a key role in drug resistance of leukaemia cells remains unclear. The present study systematically investigated aerobic glycolytic alterations and regulation in K562/adriamycin (ADM) multidrug‑resistant (MDR) and ADM‑sensitive K562 leukaemia cells in normoxia, and the association between drug resistance and improper glycometabolism. The cell proliferating activity was assessed with an MTT colorimetric assay, glycolysis, including glucose consumption, lactate export and key‑enzyme activity was determined by corresponding commercial testing kits. The expression levels of hexokinase‑II (HK‑II), lactate dehydrogenase A (LDHA), glucose transporter‑4 (GLUT‑4), AKT, p‑AKT473/308, mammalian target of rapamycin (mTOR), p‑mTOR, c‑Myc and hypoxia‑inducible factor‑1α (HIF‑1α) were analyzed by western blot or reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). K562/ADM cells exhibited increased glucose consumption and lactate accumulation, increased lactate dehydrogenase, hexokinase and pyruvate kinase activities, and reduced phosphofructokinase activity. In addition, K562/ADM cells expressed significantly more HK‑II and GLUT‑4. Notably, inhibition of glycolysis effectively killed sensitive and resistant leukaemia cells and potently restored the sensitivity of MDR cells to the anticancer agent ADM. The AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (mTOR) signalling pathway, a crucial regulator of glycometabolic homeostasis, mediated over‑activation and upregulation of c‑Myc expression levels in K562/ADM cells, which directly stimulated glucose consumption and enhanced glycolysis. In conclusion, the present

Genome Editing of Erythroid Cell Culture Model Systems.

PubMed

Yik, Jinfen J; Crossley, Merlin; Quinlan, Kate G R

2018-01-01

Genome editing to introduce specific mutations or to knock out genes in model cell systems has become an efficient platform for research in the fields of molecular biology, genetics, and cell biology. With recent rapid improvements in genome editing techniques, bench-top manipulation of the genome in cell culture has become progressively easier. The application of this knowledge to erythroid cell culture systems now allows the rapid analysis of the downstream effects of virtually any engineered gene disruption or modification in cell systems. Here, we describe a CRISPR/Cas9-based approach to making genomic modifications in erythroid lineage cells which we have successfully used in both murine (MEL) and human (K562) erythroleukaemia immortalized cell lines.

20(S)-Ginsenoside Rh2 Induce the Apoptosis and Autophagy in U937 and K562 Cells.

PubMed

Zhuang, Jianjian; Yin, Juxin; Xu, Chaojian; Mu, Ying; Lv, Shaowu

2018-03-08

Acute myeloid leukemia (AML) and Chronic myelogenous leukemia (CML) are common leukemia in adults. 20(S)-GRh2 is an important bioactive substance that is present in Panax ginseng. However, there are no investigations that deal with the comparison of apoptosis, the occurrence of autophagy, and the relationship between apoptosis and autophagy after being treated with 20(S)-GRh2 in AML and CML. In this study, we explored the effect of 20(S)-GRh2 on the AML and CML (U937 and K562). Fluorescence microscopy, CCK-8, Quantitative realtime PCR, Western blot, transmission electron microscopy (TEM), and flow cytometric analysis were used to detect the occurrence of cell proliferation inhibition, apoptosis, and autophagy. By using the above methods, it was determined that apoptosis induced by 20(S)-GRh2 was more obvious in K562 than U937 cells and 20(S)-GRh2 could generate autophagy in K562 and U937 cells. When pretreated by a specific inhibitor of autophagy, (3-methyladenine), the 20(S)-GRh2-induced apoptosis was enhanced, which indicated that 20(S)-GRh2-induced autophagy may protect U937 and K562 cells from undergoing apoptotic cell death. On the other hand, pretreated by an apoptosis suppressor (Z-VAD-FMK), it greatly induced the autophagy and partially prevented 20(S)-GRh2 induced apoptosis. This phenomenon indicated that 20(S)-GRh2-induced autophagy may serve as a survival mechanism and apoptosis and autophagy could act as partners to induce cell death in a cooperative manner. These findings may provide a rationale for future clinical application by using 20(S)-GRh2 combined autophagy inhibitors for AML and CML.

Differentiation of K562 cells under ELF-EMF applied at different time courses.

PubMed

Ayşe, Inhan-Garip; Zafer, Akan; Sule, Oncul; Işil, Işal-Turgut; Kalkan, Tunaya

2010-08-01

The time-course of ELF-EMF application to biological systems is thought to be an important parameter determining the physiological outcome. This study investigated the effect of ELF-EMF on the differentiation of K562 cells at different time courses. ELF-EMF (50 Hz, 5 mT, 1 h) was applied at two different time-courses; first at the onset of hemin induction for 1 h, and second, daily 1 h for four days. While single exposure to ELF-EMF resulted in a decrease in differentiation, ELF-EMF applied everyday for 1 h caused an increase in differentiation. The effect of co-stressors, magnesium, and heat-shock was also determined and similar results were obtained. ELF-EMF increased ROS levels in K562 cells not treated with hemin, however did not change ROS levels of hemin treated cells indicating that ROS was not the cause. Overall, these results imply that the time-course of application is an important parameter determining the physiological response of cells to ELF-EMF.

Cytotoxic and Apoptotic Effects of Different Extracts of Artemisia turanica Krasch. on K562 and HL-60 Cell Lines

PubMed Central

Tayarani-Najaran, Zahra; Sareban, Mahla; Gholami, Atefeh; Emami, Seyed Ahmad; Mojarrab, Mahdi

2013-01-01

Artemisia is an important genus of Iranian flora. Cytotoxic activities for some species of the genus have already been reported. In this study, we have investigated the cytotoxic effects of n-hexane, CH2Cl2, EtOAc, EtOH, and EtOH/H2O (1 : 1) extracts of A. turanica Krasch. on two human leukemic cancer cell lines (K562 and HL-60) and J774 as normal cells using alamarBlue (resazurin) assay. PI staining of the fragmented DNA and western blot analysis were used to evaluate the possible apoptotic effect of the extract. The CH2Cl2 extract of A. turanica showed the most antiproliferative effect on cancer cells among all tested extracts with IC50 values of 69 and 104 μg/mL on K562 and HL-60 cells, respectively, whereas the normal cells were not affected significantly by this extract. Sub-G1 peak in the flow cytometry histogram of the cells treated with CH2Cl2 extract of A. turanica and cleavage of PARP protein confirmed the induction of apoptosis with CH2Cl2 extract. Taken together, the findings of the present work suggest the anticancer potential of CH2Cl2 extract of A. turanica on human leukemic cancer cell lines. PMID:24288497

Apoptosis of leukemia K562 and Molt-4 cells induced by emamectin benzoate involving mitochondrial membrane potential loss and intracellular Ca2+ modulation.

PubMed

Yun, Xinming; Rao, Wenbing; Xiao, Ciying; Huang, Qingchun

2017-06-01

Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca 2+ concentration ([Ca 2+ ] i ) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10μM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48h exposure to 10μM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca 2+ ] i level was peaked and persisted within 70s in K562 cells whilst 50s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia. Copyright © 2017 Elsevier B.V. All rights reserved.

Bcr-Abl-independent mechanism of resistance to imatinib in K562 cells: Induction of cyclooxygenase-2 (COX-2) by histone deacetylases (HDACs).

PubMed

Kalle, Arunasree M; Sachchidanand, Sachchidanand; Pallu, Reddanna

2010-09-01

Our previous studies have shown that overexpression of MDR1 and cyclooygenase-2 (COX-2) resulted in resistance development to imatinib in chronic myelogenous leukemia (CML) K562 (IR-K562) cells. In the present study, the regulatory mechanism of MDR1 induction by COX-2 was investigated. A gradual overexpression of MDR1 and COX-2 during the process of development was observed. Furthermore, down regulation of MDR1 upon COX-2 knockdown by siRNA showed a decrease in the PKC levels and activation of PKC by addition of PGE(2) to K562 cells, suggesting a role for PKC in the COX-2 mediated induction of MDR1. The present study demonstrates COX-2 induction by HDACs and MDR1 induction by COX-2 via PGE(2)-cAMP-PKC-mediated pathway. Copyright 2010 Elsevier Ltd. All rights reserved.

Analysis of the Effects of δ-Tocopherol on RAW264.7 and K562 Cells Based on 1H NMR Metabonomics.

PubMed

Lu, Yang; Li, Hui; Geng, Yue

2018-01-31

δ-Tocopherol (δ-TOH) is a form of vitamin E with higher bioactivity. In this study, we studied the bioactivity of δ-TOH using the IC 50 of δ-TOH on RAW264.7 (80 μM) and K562 (110 μM) cells. We compared the differential metabolites from the cell lines with and without δ-TOH treatment by 1 H NMR metabonomics analysis. It was found that δ-TOH affected the protein biosynthesis, betaine metabolism, and urea cycle in various ways in both cell lines. Metabolic levels of the cell lines were changed after treatment with δ-TOH as differential metabolites were produced. The betaine level in RAW264.7 cells was reduced significantly, while the l-lactic acid level in K562 cells was significantly enhanced. The metabolic changes might contribute to the switch of the respiration pattern from aerobic respiration to anaerobic respiration in K562 cells. These results are helpful in further understanding the subtoxicity of δ-TOH.

Granulocyte-macrophage colony-stimulating factor induces the differentiation of murine erythroleukaemia cells into dendritic cells.

PubMed Central

Cao, X; Zhao, Y; Yu, Y; Wang, Y; Zhang, M; Zhang, W; Wang, J

1998-01-01

Dendritic cells (DC) are professional antigen-presenting cells (APC) within the immune system and antigen-pulsed DC can be used as an effective vaccine for active immunotherapy of cancer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the generation of DC. We previously showed that GM-CSF can induce murine erythroleukaemia cells (FBL-3) to differentiate into monocyte-like cells. To develop a new vaccinating method to stimulate the host immune response to leukaemia, we further investigate whether FBL-3 cells induced by GM-CSF can differentiate into DC in the present study. After being treated with GM-CSF, FBL-3 cells expressed high levels of 33D1 and NLDC-145, which are the specific markers of DC. The expression of MHC-II, B7-1, B7-2 and vascular cell adhesion molecule-1 (VCAM-1) was up-regulated markedly; the typical morphology of DC were also observed by electron microscopy. Functionally, the GM-CSF-induced FBL-3 cells could apparently stimulate the proliferation of naive allogeneic and autologous T lymphocytes and induce the generation of specific CTL more efficiently than the wild-type FBL-3 cells. Mice immunized with GM-CSF-induced FBL-3 cells could resist the subsequent challenge with the wild-type FBL-3 cells. Collectively, these data indicate that GM-CSF differentiates murine erythroleukaemia cells into DC phenotypically, morphologically and functionally. FBL-3-derived DC can be used as a new type of vaccine. Our results may have important implications for the immunotherapy of leukaemia. Images Figure 3 Figure 4 PMID:9767469

Differential chemosensitization of P-glycoprotein overexpressing K562/Adr cells by withaferin A and Siamois polyphenols

PubMed Central

2010-01-01

Background Multidrug resistance (MDR) is a major obstacle in cancer treatment and is often the result of overexpression of the drug efflux protein, P-glycoprotein (P-gp), as a consequence of hyperactivation of NFκB, AP1 and Nrf2 transcription factors. In addition to effluxing chemotherapeutic drugs, P-gp also plays a specific role in blocking caspase-dependent apoptotic pathways. One feature that cytotoxic treatments of cancer have in common is activation of the transcription factor NFκB, which regulates inflammation, cell survival and P-gp expression and suppresses the apoptotic potential of chemotherapeutic agents. As such, NFκB inhibitors may promote apoptosis in cancer cells and could be used to overcome resistance to chemotherapeutic agents. Results Although the natural withanolide withaferin A and polyphenol quercetin, show comparable inhibition of NFκB target genes (involved in inflammation, angiogenesis, cell cycle, metastasis, anti-apoptosis and multidrug resistance) in doxorubicin-sensitive K562 and -resistant K562/Adr cells, only withaferin A can overcome attenuated caspase activation and apoptosis in K562/Adr cells, whereas quercetin-dependent caspase activation and apoptosis is delayed only. Interestingly, although withaferin A and quercetin treatments both decrease intracellular protein levels of Bcl2, Bim and P-Bad, only withaferin A decreases protein levels of cytoskeletal tubulin, concomitantly with potent PARP cleavage, caspase 3 activation and apoptosis, at least in part via a direct thiol oxidation mechanism. Conclusions This demonstrates that different classes of natural NFκB inhibitors can show different chemosensitizing effects in P-gp overexpressing cancer cells with impaired caspase activation and attenuated apoptosis. PMID:20438634

Regulation of HtrA2 on WT1 gene expression under imatinib stimulation and its effects on the cell biology of K562 cells.

PubMed

Zhang, Lixia; Li, Yan; Li, Xiaoyan; Zhang, Qing; Qiu, Shaowei; Zhang, Qi; Wang, Min; Xing, Haiyan; Rao, Qing; Tian, Zheng; Tang, Kejing; Wang, Jianxiang; Mi, Yingchang

2017-09-01

The aim of the present study was to investigate the regulation of Wilms Tumor 1 (WT1) by serine protease high-temperature requirement protein A2 (HtrA2), a member of the Htr family, in K562 cells. In addition, the study aimed to observe the effect of this regulation on cell biological functions and its associated mechanisms. Expression of WT1 and HtrA2 mRNA, and proteins following imatinib and the HtrA2 inhibitor 5-[5-(2-nitrophenyl) furfuryl iodine]-1, 3-diphenyl-2-thiobarbituric acid (UCF-101) treatment was detected with reverse transcription-quantitative polymerase chain reaction and western blot analysis. Subsequent to treatment with drugs and UCF-101, the proliferative function of K562 cells was detected using MTT assays, and the rate of apoptosis was detected using Annexin V with propidium iodide flow cytometry in K562 cells. The protein levels in the signaling pathway were analyzed using western blotting following treatment with imatinib and UCF-101. In K562 cells, imatinib treatment activated HtrA2 gene at a transcription level, while the WT1 gene was simultaneously downregulated. Following HtrA2 inhibitor (UCF-101) treatment, the downregulation of WT1 increased gradually. At the protein level, imatinib induced the increase in HtrA2 protein level and concomitantly downregulated WT1 protein level. Subsequent to HtrA2 inhibition by UCF-101, the WT1 protein level decreased temporarily, but eventually increased. Imatinib induced apoptosis in K562 cells, but this effect was attenuated by the HtrA2 inhibitor UCF-101, resulting in the upregulation of the WT1 protein level. However; UCF-101 did not markedly change the proliferation inhibition caused by imatinib. Imatinib activated the p38 mitogen activated protein kinase (p38 MAPK) signaling pathway in K562 cells, and UCF-101 affected the activation of imatinib in the p38 MAPK signaling pathway. Imatinib inhibited the extracellular signal-related kinase (ERK1/2) pathway markedly and persistently, but UCF-101

Jellyfish extract induces apoptotic cell death through the p38 pathway and cell cycle arrest in chronic myelogenous leukemia K562 cells

PubMed Central

Kwak, Choong-Hwan; Abekura, Fukushi; Park, Jun-Young; Park, Nam Gyu; Chang, Young-Chae; Lee, Young-Choon; Chung, Tae-Wook; Ha, Ki-Tae; Son, Jong-Keun

2017-01-01

Jellyfish species are widely distributed in the world’s oceans, and their population is rapidly increasing. Jellyfish extracts have several biological functions, such as cytotoxic, anti-microbial, and antioxidant activities in cells and organisms. However, the anti-cancer effect of Jellyfish extract has not yet been examined. We used chronic myelogenous leukemia K562 cells to evaluate the mechanisms of anti-cancer activity of hexane extracts from Nomura’s jellyfish in vitro. In this study, jellyfish are subjected to hexane extraction, and the extract is shown to have an anticancer effect on chronic myelogenous leukemia K562 cells. Interestingly, the present results show that jellyfish hexane extract (Jellyfish-HE) induces apoptosis in a dose- and time-dependent manner. To identify the mechanism(s) underlying Jellyfish-HE-induced apoptosis in K562 cells, we examined the effects of Jellyfish-HE on activation of caspase and mitogen-activated protein kinases (MAPKs), which are responsible for cell cycle progression. Induction of apoptosis by Jellyfish-HE occurred through the activation of caspases-3,-8 and -9 and phosphorylation of p38. Jellyfish-HE-induced apoptosis was blocked by a caspase inhibitor, Z-VAD. Moreover, during apoptosis in K562 cells, p38 MAPK was inhibited by pretreatment with SB203580, an inhibitor of p38. SB203580 blocked jellyfish-HE-induced apoptosis. Additionally, Jellyfish-HE markedly arrests the cell cycle in the G0/G1 phase. Therefore, taken together, the results imply that the anti-cancer activity of Jellyfish-HE may be mediated apoptosis by induction of caspases and activation of MAPK, especially phosphorylation of p38, and cell cycle arrest at the Go/G1 phase in K562 cells. PMID:28133573

Allium Roseum L. Extract Exerts Potent Suppressive Activities on Chronic Myeloid Leukemia K562 Cell Viability Through the Inhibition of BCR-ABL, PI3K/Akt, and ERK1/2 Pathways and the Abrogation of VEGF Secretion.

PubMed

Souid, Soumaya; Najjaa, Hanen; Riahi-Chebbi, Ichrak; Haoues, Meriam; Neffati, Mohamed; Arnault, Ingrid; Auger, Jacques; Karoui, Habib; Essafi, Makram; Essafi-Benkhadir, Khadija

2017-01-01

Use of plant extracts, alone or combined to the current chemotherapy as chemosensitizers, has emerged as a promising strategy to overcome tumor drug resistance. Here, we investigated the anticancer activity of Allium roseum L. extracts, a wild edible species in North Africa, on human Chronic Myeloid Leukemia (CML) K562 cells. The dehydrated aqueous extract (DAE) disturbed the cell cycle progression and induced the apoptosis of K562 cells. Chemical analysis of DAE showed a diversity of organosulfur compounds S-alk(en)yl-cysteine sulfoxides (RCSO) and high amount of allicin, suggesting that such molecule may be behind its antitumor effect. DAE was efficient in inhibiting K562 cell viability. DAE inhibitory effect was associated with the dephosphorylation of the BCR-ABL kinase and interfered with ERK 1/2 , Akt, and STAT5 pathways. Furthermore, we found that DAE-induced inactivation of Akt kinase led to the activation of its target FOXO3 transcription factor, enhancing the expression of FOXO3-regulated proapoptotic effectors, Bim and Bax, and cell cycle inhibitor p27. Finally, we found that DAE reduced the secretion of vascular endothelial growth factor. Overall, our data suggest that A. roseum extract has great potential as a nontoxic cheap and effective alternative to conventional chemotherapy.

A new disposable electrode for electrochemical study of leukemia K562 cells and anticancer drug sensitivity test.

PubMed

Yu, Chunmei; Zhu, Zhenkun; Wang, Li; Wang, Qiuhong; Bao, Ning; Gu, Haiying

2014-03-15

Developing cost-effective and simple analysis tools is of vital importance for practical applications in bioanalysis. In this work, a new disposable electrochemical cell sensor with low cost and simple fabrication was proposed to study the electrochemical behavior of leukemia K562 cells and the effect of anticancer drugs on cell viability. The analytical device was integrated by using ITO glass as the substrate of working electrodes and paper as the electrolytic cell. The cyclic voltammetry of the K562 cells at the disposable electrode exhibited an irreversible anodic peak and the peak current is proportional to the cell number. This anodic peak is attributed to the oxidation of guanine in cells involving two protons per transfer of two electrons. For the drug sensitivity tests, arsenic trioxide and cyclophosphamide were added to cell culture media. As a result, the electrochemical responses of the K562 cells decreased significantly. The cytotoxicity curves and results obtained corresponded well with the results of CCK-8 assays. In comparison to conventional methods, the proposed method is simple, rapid and inexpensive. More importantly, the developed sensor is supposed to be a single-use disposable device and electrodes were prepared "as new" for each experiment. We think that such disposable electrodes with these characteristics are suitable for experimental study with cancer cells or other types of pathogens for disease diagnosis, drug selection and on-site monitoring. © 2013 Elsevier B.V. All rights reserved.

Involvement of PKC and ROS in the cytotoxic mechanism of anti-leukemic decursin and its derivatives and their structure-activity relationship in human K562 erythroleukemia and U937 myeloleukemia cells.

PubMed

Kim, Hyeon Ho; Sik Bang, Sung; Seok Choi, Jin; Han, Hogyu; Kim, Ik-Hwan

2005-06-08

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Recently, various PKC modulators were used as a chemotherapeutic agent of leukemia. Decursin (1), a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. For the development of more effective anticancer agents with PKC modulation activity, 11 decursin derivatives 2-12 were chemically synthesized and evaluated for their ability to act as a tumor-suppressing PKC activator and as an antagonist to phorbol 12-myristate 13-acetate (PMA), a tumor-promoting PKC activator. In the presence of phosphatidylserine (PS), all of 12 compounds 1-12 activated PKC (mainly alpha, beta, and gamma isozymes) but only three compounds 1-3 activated PKC even in the absence of PS. Six compounds 1-6 containing the coumarin structure were cytotoxic to human K562 erythroleukemia and U937 myeloleukemia cells. A cytotoxic mechanism of decursin and its derivatives was investigated using TUR cells, a PKC betaII-deficient variant of U937 cells. Among six compounds 1-6 with cytotoxicity to K562 and U937 leukemia cells, only three compounds 1-3 were cytotoxic to TUR cells. Therefore, compounds 1-3 and 4-6 inhibit the proliferation of leukemia cells in a PKC betaII-independent and dependent manner, respectively, indicating that the side chain of compounds determines the dependency of their cytotoxicity on PKC betaII. To further elucidate the cytotoxic mechanism of compounds 1 and 2, levels of PKC isozymes and generation of reactive oxygen species (ROS) were investigated. Compounds 1-2 induced the down-regulation of PKC alpha and betaII in K562 cells and the production of ROS in U937 cells. Thus, PKC and ROS are probably important factors in the cytotoxic mechanism of compounds 1-2. From these results, the structure-activity relationship of decursin and its derivatives

Bardoxolone methyl (CDDO-Me or RTA402) induces cell cycle arrest, apoptosis and autophagy via PI3K/Akt/mTOR and p38 MAPK/Erk1/2 signaling pathways in K562 cells.

PubMed

Wang, Xin-Yu; Zhang, Xue-Hong; Peng, Li; Liu, Zheng; Yang, Yin-Xue; He, Zhi-Xu; Dang, Hong-Wan; Zhou, Shu-Feng

2017-01-01

Chronic myeloid leukemia (CML) treatment remains a challenge due to drug resistance and severe side effect, rendering the need on the development of novel therapeutics. CDDO-Me (Bardoxolone methyl), a potent Nrf2 activator and NF-κB inhibitor, is a promising candidate for cancer treatment including leukemia. However, the underlying mechanism for CDDO-Me in CML treatment is unclear. This study aimed to evaluate the molecular interactome of CDDO-Me in K562 cells using the quantitative proteomics approach stable-isotope labeling by amino acids in cell culture (SILAC) and explore the underlying mechanisms using cell-based functional assays. A total of 1,555 proteins responded to CDDO-Me exposure, including FANCI, SRPK2, XPO5, HP1BP3, NELFCD, Na + ,K + -ATPase 1, etc. in K562 cells. A total of 246 signaling pathways and 25 networks regulating cell survival and death, cellular function and maintenance, energy production, protein synthesis, response to oxidative stress, and nucleic acid metabolism were involved. Our verification experiments confirmed that CDDO-Me down-regulated Na + ,K + -ATPase α1 in K562 cells, and significantly arrested cells in G 2 /M and S phases, accompanied by remarkable alterations in the expression of key cell cycle regulators. CDDO-Me caused mitochondria-, death receptor-dependent and ER stress-mediated apoptosis in K562 cells, also induced autophagy with the suppression of PI3K/Akt/mTOR signaling pathway. p38 MAPK/Erk1/2 signaling pathways contributed to both apoptosis- and autophagy-inducing effects of CDDO-Me in K562 cells. Taken together, these data demonstrate that CDDO-Me is a potential anti-cancer agent that targets cell cycle, apoptosis, and autophagy in the treatment of CML.

Bardoxolone methyl (CDDO-Me or RTA402) induces cell cycle arrest, apoptosis and autophagy via PI3K/Akt/mTOR and p38 MAPK/Erk1/2 signaling pathways in K562 cells

PubMed Central

Wang, Xin-Yu; Zhang, Xue-Hong; Peng, Li; Liu, Zheng; Yang, Yin-Xue; He, Zhi-Xu; Dang, Hong-Wan; Zhou, Shu-Feng

2017-01-01

Chronic myeloid leukemia (CML) treatment remains a challenge due to drug resistance and severe side effect, rendering the need on the development of novel therapeutics. CDDO-Me (Bardoxolone methyl), a potent Nrf2 activator and NF-κB inhibitor, is a promising candidate for cancer treatment including leukemia. However, the underlying mechanism for CDDO-Me in CML treatment is unclear. This study aimed to evaluate the molecular interactome of CDDO-Me in K562 cells using the quantitative proteomics approach stable-isotope labeling by amino acids in cell culture (SILAC) and explore the underlying mechanisms using cell-based functional assays. A total of 1,555 proteins responded to CDDO-Me exposure, including FANCI, SRPK2, XPO5, HP1BP3, NELFCD, Na+,K+-ATPase 1, etc. in K562 cells. A total of 246 signaling pathways and 25 networks regulating cell survival and death, cellular function and maintenance, energy production, protein synthesis, response to oxidative stress, and nucleic acid metabolism were involved. Our verification experiments confirmed that CDDO-Me down-regulated Na+,K+-ATPase α1 in K562 cells, and significantly arrested cells in G2/M and S phases, accompanied by remarkable alterations in the expression of key cell cycle regulators. CDDO-Me caused mitochondria-, death receptor-dependent and ER stress-mediated apoptosis in K562 cells, also induced autophagy with the suppression of PI3K/Akt/mTOR signaling pathway. p38 MAPK/Erk1/2 signaling pathways contributed to both apoptosis- and autophagy-inducing effects of CDDO-Me in K562 cells. Taken together, these data demonstrate that CDDO-Me is a potential anti-cancer agent that targets cell cycle, apoptosis, and autophagy in the treatment of CML. PMID:29118925

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

PubMed

Chieregato, Katia; Zanon, Cristina; Castegnaro, Silvia; Bernardi, Martina; Amati, Eliana; Sella, Sabrina; Rodeghiero, Francesco; Astori, Giuseppe

2017-01-01

Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

Comparative Study of Different Nano-Formulations of Curcumin for Reversal of Doxorubicin Resistance in K562R Cells.

PubMed

Dash, Tapan K; Konkimalla, V Badireenath

2017-02-01

Curcumin is very well established as a chemo-therapeutic, chemo-preventive and chemo-sensitizing agent in diverse disease conditions. As the isolated pure form has poor solubility and pharmacokinetic problems, therefore it is encapsulated in to several nano-formulations to improve its bioavailability. Here in the current study, we aim to compare different nano-formulations of curcumin for their chemo-sensitizing activity in doxorubicin (DOX) resistant K562 cells. Four different curcumin formulations were prepared namely DMSO assisted curcumin nano-dispersion (CurD, 260 nm), liposomal curcumin (CurL, 165 nm), MPEG-PCL micellar curcumin (CurM, 18 nm) and cyclodextrin encapsulated curcumin (CurN, 37 nm). The formulations were subjected to particle characterizations (size, zeta potential, release studies), followed by biological assays such as cellular uptake, P-gp inhibitory activity and reversal of DOX resistance by co-treatment with DOX. Curcumin uptake in K562N and K562R cells was mildly reduced when treated with CurL and CurM, while for CurD and CurN the uptake remained equivalent. However, CurL retained P-gp inhibitory activity of curcumin and with a considerable chemo-sensitizing effect but CurM showed no P-gp inhibitory activity. CurN retained above biological activities, but requires a secondary carrier under in vivo conditions. From the results, CurM was found to be most suitable for solubilization of curcumin where as CurL can be considered as most suitable nano-formulation for reversal of DOX resistance.

Thiosemicarbazone p-Substituted Acetophenone Derivatives Promote the Loss of Mitochondrial Δψ, GSH Depletion, and Death in K562 Cells

PubMed Central

Pessoto, Felipe S.; Yokomizo, Cesar H.; Prieto, Tatiana; Fernandes, Cleverton S.; Silva, Alan P.; Kaiser, Carlos R.; Basso, Ernani A.; Nantes, Iseli L.

2015-01-01

A series of thiosemicarbazone (TSC) p-substituted acetophenone derivatives were synthesized and chemically characterized. The p-substituents appended to the phenyl group of the TSC structures were hydrogen, fluor, chlorine, methyl, and nitro, producing compounds named TSC-H, TSC-F, TSC-Cl, TSC-Me, and TSC-NO2, respectively. The TSC compounds were evaluated for their capacity to induce mitochondrial permeability, to deplete mitochondrial thiol content, and to promote cell death in the K562 cell lineage using flow cytometry and fluorescence microscopy. TSC-H, TSC-F, and TSC-Cl exhibited a bell-shaped dose-response curve for the induction of apoptosis in K562 cells due to the change from apoptosis to necrosis as the principal mechanism of cell death at the highest tested doses. TSC-Me and TSC-NO2 exhibited a typical dose-response profile, with a half maximal effective concentration of approximately 10 µM for cell death. Cell death was also evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed lower toxicity of these compounds for peripheral blood mononuclear cells than for K562 cells. The possible mechanisms leading to cell death are discussed based on the observed effects of the new TSC compounds on the cellular thiol content and on mitochondrial bioenergetics. PMID:26075034

Induction of apoptosis in K562 cells by dicyclohexylammonium salt of hyperforin through a mitochondrial-related pathway.

PubMed

Liu, Jin-Yun; Liu, Zhong; Wang, Dong-Mei; Li, Man-Mei; Wang, Shao-Xiang; Wang, Rui; Chen, Jian-Ping; Wang, Yi-Fei; Yang, De-Po

2011-04-25

Hyperforin is an abundant phloroglucinol-type constituent isolated from the extract of the flowering upper portion of the plant Hypericum perforatum L. The dicyclohexylammonium salt of hyperforin (DCHA-HF) has exhibited antitumor and antiangiogenic activities in various cancer cells. Here, the antitumor effects of DCHA-HF on the chronic myeloid leukemia K562 cell line were investigated for the first time. DCHA-HF exhibited dose- and time-dependent inhibitory activities against K562 cells, with IC(50) values of 8.6 and 3.2 μM for 48 h and 72 h of treatment, respectively, which was more effective than that of the hyperforin. In contrast, little cytotoxic activity was observed with DCHA-HF on HUVECs. DCHA-HF treatment resulted in induction of apoptosis as evidenced from DNA fragmentation, nuclear condensation and increase of early apoptotic cells by DAPI staining analysis, TUNEL assay and Annexin V-FITC/PI double-labeled staining analysis, respectively. Moreover, DCHA-HF elicited dissipation of mitochondrial transmembrane potential that commenced with the release of cytochrome c through down-regulation of expression of anti-apoptotic proteins and up-regulation of expression of pro-apoptotic proteins. DCHA-HF treatment induced activation of the caspase 3, 8, and 9 cascade and subsequent PARP cleavage, and DCHA-HF-induced apoptosis was significantly inhibited by caspase inhibitors. Treated cells were arrested at the G1 phase of the cell cycle and the expression of p53 and p27(Kip1), two key regulators related to cell cycle and apoptosis, was up-regulated. These results suggest that DCHA-HF inhibits K562 cell growth by inducing caspase-dependent apoptosis mediated by a mitochondrial pathway and arresting the cell cycle at the G1 phase. Therefore, DCHA-HF is a potential chemotherapeutic antitumor drug for chronic myeloid leukemia therapy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

PubMed

Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

2015-01-01

In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. © 2015 by the Association of Clinical Scientists, Inc.

Induced apoptosis by mild hyperthermia occurs via telomerase inhibition on the three human myeloid leukemia cell lines: TF-1, K562, and HL-60.

PubMed

Deezagi, Abdolkhaleg; Manteghi, Sanaz; Khosravani, Pardis; Vaseli-Hagh, Neda; Soheili, Zahra-Soheila

2009-09-01

The purpose of this research was to understand the effect of hyperthermia on the telomerase activity in human leukemic cell lines (HL-60, K562, and TF-1). The cells were treated by hyperthermia at the range of 41-44 degrees C for 120 min and incubated for 96 h. Then telomerase activity, cell proliferation, and apoptosis were assessed. The results indicated that hyperthermia significantly induced apoptosis on the cells. The cells exhibited pre-apoptotic pattern at 41 and 42 degrees C at 60-120 min and apoptotic pattern at 43 and 44 degrees C over 30 min after hyperthermia. Telomerase activity (that was assayed immediately after hyperthermia) was stable at 41-42 degrees C for 60 min but decreased to 35-40% at 120 min. However, at severe hyperthermia (43-44 degrees C) telomerase activity was decreased in a time- and dose-dependent manner. Following hyperthermia (41-44 degrees C up to 120 min), the cells were incubated for 96 h. In these conditions, the telomerase activity was decreased by about 60-80% in comparison with that untreated control cells.

Anti-Tumor Activity of Eurycoma longifolia Root Extracts against K-562 Cell Line: In Vitro and In Vivo Study

PubMed Central

Majid, Amin Malik Shah Abdul; Kit-Lam, Chan; Abdullah, Wan Zaidah; Zaki, Abdelhamid; Jamal Din, Shah Kamal Khan; Yusoff, Narazah Mohd

2014-01-01

Eurycoma longifolia Jack has been widely used in traditional medicine for its antimalarial, aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer activity has also been recently reported on different solid tumors, however no anti-leukemic activity of this plant has been reported. Thus the present study assesses the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. The K-562 cells (purchased from ATCC) were isolated from patients with chronic myelocytic leukemia (CML) were treated with the various fractions (TAF273, F3 and F4) of E. longifolia root methanolic extract at various concentrations and time intervals and the anti-proliferative activity assessed by MTS assay. Flow cytometry was used to assess the apoptosis and cell cycle arrest. Nude mice injected subcutaneously with 107 K-562 cells were used to study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4 showed various degrees of growth inhibition with IC50 values of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose and time dependent manner. TAF273 arrested cell cycle at G1and S phases. Intraperitoneal administration of TAF273 (50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor in TAF273-treated mice compared with the control mice (P = 0.024). TAF273 shows potent anti-proliferative activity in vitro and in vivo models of CML and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management. PMID:24409284

Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates.

PubMed

Shinkai, Yasuhiro; Kashihara, Shinichi; Minematsu, Go; Fujii, Hirofumi; Naemura, Madoka; Kotake, Yojiro; Morita, Yasutaka; Ohnuki, Koichiro; Fokina, Alesya A; Stetsenko, Dmitry A; Filichev, Vyacheslav V; Fujii, Masayuki

2017-06-01

Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideβ7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideβ7 is a promising candidate for in vivo use and therapeutic applications.

Vaccination with autologous myeloblasts admixed with GM-K562 cells in patients with advanced MDS or AML after allogeneic HSCT

PubMed Central

Kim, Haesook T.; Bavli, Natalie; Mihm, Martin; Pozdnyakova, Olga; Piesche, Matthias; Daley, Heather; Reynolds, Carol; Souders, Nicholas C.; Cutler, Corey; Koreth, John; Alyea, Edwin P.; Antin, Joseph H.; Ritz, Jerome; Dranoff, Glenn; Soiffer, Robert J.

2017-01-01

We report a clinical trial testing vaccination of autologous myeloblasts admixed with granulocyte-macrophage colony-stimulating factor secreting K562 cells after allogeneic hematopoietic stem cell transplantation (HSCT). Patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with ≥5% marrow blasts underwent myeloblast collection before HSCT. At approximately day +30, 6 vaccines composed of irradiated autologous myeloblasts mixed with GM-K562 were administered. Tacrolimus-based graft-versus-host disease (GVHD) prophylaxis was not tapered until vaccine completion (∼day 100). Thirty-three patients with AML (25) and MDS (8) enrolled, 16 (48%) had ≥5% marrow blasts at transplantation. The most common vaccine toxicity was injection site reactions. One patient developed severe eosinophilia and died of eosinophilic myocarditis. With a median follow-up of 67 months, cumulative incidence of grade 2-4 acute and chronic GVHD were 24% and 33%, respectively. Relapse and nonrelapse mortality were 48% and 9%, respectively. Progression-free survival (PFS) and overall survival (OS) at 5 years were 39% and 39%. Vaccinated patients who were transplanted with active disease (≥5% marrow blasts) had similar OS and PFS at 5 years compared with vaccinated patients transplanted with <5% marrow blasts (OS, 44% vs 35%, respectively, P = .81; PFS, 44% vs 35%, respectively, P = .34). Postvaccination antibody responses to angiopoietin-2 was associated with superior OS (hazard ratio [HR], 0.43; P = .031) and PFS (HR, 0.5; P = .036). Patients transplanted with active disease had more frequent angiopoeitin-2 antibody responses (62.5% vs 20%, P = .029) than those transplanted in remission. GM-K562/leukemia cell vaccination induces biologic activity, even in patients transplanted with active MDS/AML. This study is registered at www.clinicaltrials.gov as #NCT 00809250. PMID:29296875

The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562.

PubMed

Lust, Sofie; Vanhoecke, Barbara; Van Gele, Mireille; Philippé, Jan; Bracke, Marc; Offner, Fritz

2010-06-01

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

Small Molecule TH-39 Potentially Targets Hec1/Nek2 Interaction and Exhibits Antitumor Efficacy in K562 Cells via G0/G1 Cell Cycle Arrest and Apoptosis Induction.

PubMed

Zhu, Yongxia; Wei, Wei; Ye, Tinghong; Liu, Zhihao; Liu, Li; Luo, Yong; Zhang, Lidan; Gao, Chao; Wang, Ningyu; Yu, Luoting

2016-01-01

Cancer is still a major public health issue worldwide, and new therapeutics with anti-tumor activity are still urgently needed. The anti-tumor activity of TH-39, which shows potent anti-proliferative activity against K562 cells with an IC50 of 0.78 µM, was investigated using immunoblot, co-immunoprecipitation, the MTT assay, and flow cytometry. Mechanistically, TH-39 may disrupt the interaction between Hec1 and Nek2 in K562 cells. Moreover, TH-39 inhibited cell proliferation in a concentration- and time-dependent manner by influencing the morphology of K562 cells and inducing G0/G1 phase arrest. G0/G1 phase arrest was associated with down-regulation of CDK2-cyclin E complex and CDK4/6-cyclin D complex activities. Furthermore, TH-39 also induced cell apoptosis, which was associated with activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax. TH-39 could also decrease mitochondrial membrane potential (Δψm) and increase reactive oxygen species (ROS) accumulation in K562 cells. The results indicated that TH-39 might induce apoptosis via the ROS-mitochondrial apoptotic pathway. This study highlights the potential therapeutic efficacy of the anti-cancer compound TH-39 in treatment-resistant chronic myeloid leukemia. © 2016 The Author(s) Published by S. Karger AG, Basel.

4-Nerolidylcatechol: apoptosis by mitochondrial mechanisms with reduction in cyclin D1 at G0/G1 stage of the chronic myelogenous K562 cell line.

PubMed

Benfica, Polyana Lopes; Ávila, Renato Ivan de; Rodrigues, Bruna Dos Santos; Cortez, Alane Pereira; Batista, Aline Carvalho; Gaeti, Marilisa Pedroso Nogueira; Lima, Eliana Martins; Rezende, Kênnia Rocha; Valadares, Marize Campos

2017-12-01

4-Nerolidylcatechol (4-NRC) has showed antitumor potential through apoptosis. However, its apoptotic mechanisms are still unclear, especially in leukemic cells. To evaluate the cytotoxic potential of 4-NRC and its cell death pathways in p53-null K562 leukemic cells. Cytotoxicity of 4-NRC (4.17-534.5 μM) over 24 h of exposure was evaluated by MTT assay. 4-NRC-induced apoptosis in K562 cells was investigated by phosphatidylserine (PS) externalization, cell cycle, sub-G1, mitochondrial evaluation, cytochrome c, cyclin D1 and intracellular reactive oxygen species (ROS) levels, and caspase activity analysis. IC 50 values obtained were 11.40, 27.31, 15.93 and 15.70 μM for lymphocytes, K562, HL-60 and Jurkat cells, respectively. In K562 cells, 4-NRC (27 μM) promoted apoptosis as verified by cellular morphological changes, a significant increase in PS externalization and sub-G1 cells. Moreover, it significantly arrested the cells at the G0/G1 phase due to a reduction in cyclin D1 expression. These effects of 4-NRC also significantly promoted a reduction in mitochondrial activity and membrane depolarization, accumulation of cytosolic cytochrome c and ROS overproduction. Additionally, it triggered an increase in caspases -3/7, -8 and -9 activities. When the cells were pretreated with N-acetyl-l-cysteine ROS scavenger, 4-NRC-induced apoptosis was partially blocked, which suggests that it exerts cytotoxicity though not exclusively through ROS-mediated mechanisms. 4-NRC has antileukemic properties, inducing apoptosis mediated by mitochondrial-dependent mechanisms with cyclin D1 inhibition. Given that emerging treatment concepts include novel combinations of well-known agents, 4-NRC could offer a promising alternative for chemotherapeutic combinations to maximize tumour suppression.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin

PubMed Central

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-01-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. PMID:26342260

Comparison of different methods for erythroid differentiation in the K562 cell line.

PubMed

Shariati, Laleh; Modaress, Mehran; Khanahmad, Hossein; Hejazi, Zahra; Tabatabaiefar, Mohammad Amin; Salehi, Mansoor; Modarressi, Mohammad Hossein

2016-08-01

To compare methods for erythroid differentiation of K562 cells that will be promising in the treatment of beta-thalassemia by inducing γ-globin synthesis. Cells were treated separately with: RPMI 1640 medium without glutamine, RPMI 1640 medium without glutamine supplemented with 1 mM sodium butyrate, RPMI 1640 medium supplemented with 1 mM sodium butyrate, 25 µg cisplatin/ml, 0.1 µg cytosine arabinoside/ml. The highest differentiation (84 %) with minimum toxicity was obtained with cisplatin at 15 µg /ml. Real-time RT-PCR showed that expression of the γ-globin gene was significantly higher in the cells differentiated with cisplatin compared to undifferentiated cells (P < 0.001). Cisplatin is useful in the experimental therapy of ß-globin gene defects and can be considered for examining the basic mechanism of γ-reactivation.

Targeted Blockage of Signal Transducer and Activator of Transcription 5 Signaling Pathway with Decoy Oligodeoxynucleotides Suppresses Leukemic K562 Cell Growth

PubMed Central

Wang, Xiaozhong; Zeng, Jianming; Shi, Mei; Zhao, Shiqiao; Bai, Weijun; Cao, Weixi; Tu, Zhiguang; Huang, Zonggan

2011-01-01

The protein signal transducer and activator of transcription 5 (STAT5) of the JAK/STAT pathway is constitutively activated because of its phosphorylation by tyrosine kinase activity of fusion protein BCR-ABL in chronic myelogenous leukemia (CML) cells. This study investigated the potential therapeutic effect of STAT5 decoy oligodeoxynucleotides (ODN) using leukemia K562 cells as a model. Our results showed that transfection of 21-mer-long STAT5 decoy ODN into K562 cells effectively inhibited cell proliferation and induced cell apoptosis. Further, STAT5 decoy ODN downregulated STAT5 targets bcl-xL, cyclinD1, and c-myc at both mRNA and protein levels in a sequence-specific manner. Collectively, these data demonstrate the therapeutic effect of blocking the STAT5 signal pathway by cis-element decoy for cancer characterized by constitutive STAT5 activation. Thus, our study provides support for STAT5 as a potential target downstream of BCR-ABL for CML treatment and helps establish the concept of targeting STAT5 by decoy ODN as a novel therapy approach for imatinib-resistant CML. PMID:21091189

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin.

PubMed

Finotti, Alessia; Gasparello, Jessica; Breveglieri, Giulia; Cosenza, Lucia Carmela; Montagner, Giulia; Bresciani, Alberto; Altamura, Sergio; Bianchi, Nicoletta; Martini, Elisa; Gallerani, Eleonora; Borgatti, Monica; Gambari, Roberto

2015-12-01

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of β-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the β-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

31 CFR 562.203 - Holding of funds in interest-bearing accounts; investment and reinvestment.

Code of Federal Regulations, 2013 CFR

2013-07-01

... IRANIAN HUMAN RIGHTS ABUSES SANCTIONS REGULATIONS Prohibitions § 562.203 Holding of funds in interest... accounts; investment and reinvestment. 562.203 Section 562.203 Money and Finance: Treasury Regulations... the funds are invested in a money market fund or in U.S. Treasury bills. (2) For purposes of this...

31 CFR 562.203 - Holding of funds in interest-bearing accounts; investment and reinvestment.

Code of Federal Regulations, 2012 CFR

2012-07-01

... IRANIAN HUMAN RIGHTS ABUSES SANCTIONS REGULATIONS Prohibitions § 562.203 Holding of funds in interest... accounts; investment and reinvestment. 562.203 Section 562.203 Money and Finance: Treasury Regulations... the funds are invested in a money market fund or in U.S. Treasury bills. (2) For purposes of this...

31 CFR 562.203 - Holding of funds in interest-bearing accounts; investment and reinvestment.

Code of Federal Regulations, 2014 CFR

2014-07-01

... IRANIAN HUMAN RIGHTS ABUSES SANCTIONS REGULATIONS Prohibitions § 562.203 Holding of funds in interest... accounts; investment and reinvestment. 562.203 Section 562.203 Money and Finance: Treasury Regulations... the funds are invested in a money market fund or in U.S. Treasury bills. (2) For purposes of this...

31 CFR 562.203 - Holding of funds in interest-bearing accounts; investment and reinvestment.

Code of Federal Regulations, 2011 CFR

2011-07-01

... IRANIAN HUMAN RIGHTS ABUSES SANCTIONS REGULATIONS Prohibitions § 562.203 Holding of funds in interest... accounts; investment and reinvestment. 562.203 Section 562.203 Money and Finance: Treasury Regulations... the funds are invested in a money market fund or in U.S. Treasury bills. (2) For purposes of this...

Selected terpenes from leaves of Ocimum basilicum L. induce hemoglobin accumulation in human K562 cells.

PubMed

Feriotto, Giordana; Marchetti, Nicola; Costa, Valentina; Torricelli, Piera; Beninati, Simone; Tagliati, Federico; Mischiati, Carlo

2018-06-01

Re-expression of fetal hemoglobin (HbF) was proposed as a possible therapeutic strategy for β-haemoglobinopathies. Although several inducers of HbF were tested in clinical trials, only hydroxyurea (HU) received FDA approval. Despite it produced adequate HbF levels only in half of HU-treated SCD patients, and was ineffective at all in β-thalassemia patients, beneficial effects of this approach suggested to continue in this direction identifying further molecules capable of inducing HbF. We tested the potential of essential oil isolated from Ocimum basilicum L. leaves (ObEO) in inducing hemoglobin biosynthesis. Initially, dose-dependent effect and kinetics of hemoglobin accumulation in K562 cells after treatment with ObEO were evaluated. ObEO induced dose-dependent hemoglobin accumulation superior to hydroxyurea and rapamycin and a strongest γ-globin mRNA expression. Terpenes composition of ObEO was studied by GC-MS. Three main constituents, linalool, eugenol and eucalyptol, represented about 75% of total. A blend of these three terpenes fully replicated the ObEO's biological effect, thus indicating that one of them or all together could be the active ingredients. When terpenes were tested individually, eugenol was the only one inducing stable hemoglobin accumulation, while eucalyptol and linalool produced only a small transient response. However, eugenol potential was strongly enhanced in the presence of eucalyptol and linalool, suggesting a synergistic effect on hemoglobin accumulation. By these results, the discovery of a new inducer and the interesting activity of a blend of major terpenes from ObOE on Hb accumulation could have positive fallouts on β-thalassemia and sickle cells anemia. Copyright © 2018 Elsevier B.V. All rights reserved.

32 CFR 562.6 - Responsibilities.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Responsibilities. 562.6 Section 562.6 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.6 Responsibilities. (a) The Commanding General, US Army Military Personnel Center, 200...

32 CFR 562.2 - Applicability.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Applicability. 562.2 Section 562.2 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.2 Applicability. This regulation applies to the program given at college level...

31 CFR 562.303 - Entity.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Entity. 562.303 Section 562.303 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN ASSETS... § 562.303 Entity. The term entity means a partnership, association, trust, joint venture, corporation...

32 CFR 562.1 - Purpose.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Purpose. 562.1 Section 562.1 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.1 Purpose. This regulation gives policies for conducting the Army's Senior Reserve Officers...

32 CFR 562.4 - Objectives.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Objectives. 562.4 Section 562.4 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.4 Objectives. The objectives of the ROTC program are to: (a) Attract, motivate, and prepare...

The multidrug resistance pumps are inhibited by silibinin and apoptosis induced in K562 and KCL22 leukemia cell lines.

PubMed

Noori-Daloii, Mohammad Reza; Saffari, Mojtaba; Raoofian, Reza; Yekaninejad, Mirsaeed; Dinehkabodi, Orkideh Saydi; Noori-Daloii, Ali Reza

2014-05-01

Silibinin have been introduced for several years as a potent antioxidant in the field of nutraceuticals. Based on wide persuasive effects of this drug, we have decided to investigate the effects of silibinin on chronic myelogenous leukemia (CML) in vitro models, K562 and KCL22 cell lines. Lactate dehydrogenase (LDH) release, microculture tetrazolium test (MTT assay) and real-time PCR were employed to evaluate the effects of silibinin on cell cytotoxicity, cell proliferation and expression of various multidrug resistance genes in these cell lines, respectively. Our results have shown that presence of silibinin has inhibitory effects on cell proliferation of K562 and KCL22 cell lines. Also, our data indicated that silibinin, in a dose-dependent manner with applying no cytotoxic effects, inhibited cell proliferation and reduced mRNA expression levels of some transporter genes e.g. MDR1, MRP3, MRP2, MRP1, MRP5, MRP4, ABCG2, ABCB11, MRP6 and MRP7. The multifarious in vitro inhibitory effects of silibinin are in agreement with growing body of evidence that silibinin would be an efficient anticancer agent in order to be used in multi-target therapy to prevail the therapeutic hold backs against CML. Copyright © 2014. Published by Elsevier Ltd.

32 CFR 562.3 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Definitions. 562.3 Section 562.3 National Defense... § 562.3 Definitions. The following terms apply to the Army's Senior Reserve Officers' Training Corps... installation. This is part of the advanced course and normally attended between Military Science (MS)-III and...

Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix.

PubMed

Neri, L M; Bortul, R; Zweyer, M; Tabellini, G; Borgatti, P; Marchisio, M; Bareggi, R; Capitani, S; Martelli, A M

1999-06-01

The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the

VEGFR2-targeted fusion antibody improved NK cell-mediated immunosurveillance against K562 cells.

PubMed

Ren, Xueyan; Xie, Wei; Wang, Youfu; Xu, Menghuai; Liu, Fang; Tang, Mingying; Li, Chenchen; Wang, Min; Zhang, Juan

2016-08-01

MHC class I polypeptide-related sequence A (MICA), which is normally expressed on cancer cells, activates NK cells via NK group 2-member D pathway. However, some cancer cells escape NK-mediated immune surveillance by shedding membrane MICA causing immune suppression. To address this issue, we designed an antibody-MICA fusion targeting tumor-specific antigen (vascular endothelial growth factor receptor 2, VEGFR2) based on our patented antibody (mAb04) against VEGFR2. In vitro results demonstrate that the fusion antibody retains both the antineoplastic and the immunomodulatory activity of mAb04. Further, we revealed that it enhanced NK-mediated immunosurveillance against K562 cells through increasing degranulation and cytokine production of NK cells. The overall data suggest our new fusion protein provides a promising approach for cancer-targeted immunotherapy and has prospects for potential application of chronic myeloid leukemia.

31 CFR 562.304 - Interest.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Interest. 562.304 Section 562.304... Definitions § 562.304 Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct...

31 CFR 562.304 - Interest.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Interest. 562.304 Section 562.304... Definitions § 562.304 Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct...

31 CFR 562.304 - Interest.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Interest. 562.304 Section 562.304... Definitions § 562.304 Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct...

31 CFR 562.304 - Interest.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Interest. 562.304 Section 562.304... Definitions § 562.304 Interest. Except as otherwise provided in this part, the term interest, when used with respect to property (e.g., “an interest in property”), means an interest of any nature whatsoever, direct...

Celecoxib sensitizes imatinib-resistant K562 cells to imatinib by inhibiting MRP1-5, ABCA2 and ABCG2 transporters via Wnt and Ras signaling pathways.

PubMed

Dharmapuri, Gangappa; Doneti, Ravinder; Philip, Gundala Harold; Kalle, Arunasree M

2015-07-01

Imatinib mesylate, a tyrosine kinase inhibitor, is very effective in the treatment of chronic myeloid leukemia (CML). However, development of resistance to imatinib therapy is also a very common mechanism observed with long-term administration of the drug. Our previous studies have highlighted the role of cyclooxygenase-2 (COX-2) in regulating the expression of multidrug resistant protein-1 (MDR1), P-gp, in imatinib-resistant K562 cells (IR-K562) via PGE2-cAMP-PKC-NF-κB pathway and inhibition of COX-2 by celecoxib, a COX-2 specific inhibitor, inhibits this pathway and reverses the drug resistance. Studies have identified that not only MDR1 but other ATP-binding cassette transport proteins (ABC transporters) are involved in the development of imatinib resistance. Here, we tried to study the role of COX-2 in the regulation of other ABC transporters such as MRP1, MRP2, MRP3, ABCA2 and ABCG2 that have been already implicated in imatinib resistance development. The results of the study clearly indicated that overexpression of COX-2 lead to upregulation of MRP family proteins in IR-K562 cells and celecoxib down-regulated the ABC transporters through Wnt and MEK signaling pathways. The study signifies that celecoxib in combination with the imatinib can be a good alternate treatment strategy for the reversal of imatinib resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synergistic inhibitory effects of deferasirox in combination with decitabine on leukemia cell lines SKM-1, THP-1, and K-562.

PubMed

Li, Nianyi; Chen, Qinfen; Gu, Jingwen; Li, Shuang; Zhao, Guangjie; Wang, Wei; Wang, Zhicheng; Wang, Xiaoqin

2017-05-30

A multi-center study from the French Myelodysplastic Syndrome (MDS) Group confirmed that iron chelation therapy is an independent prognostic factor that can increase the survival rate of patients who are suffering from transfusion-dependent low-risk MDS. In this study, we aimed to explore this clinical phenomena in vitro, by exploring the synergistic effect of the iron chelator Deferasirox (DFX) and the DNA methyl transferase inhibitor Decitabine (DAC) in the leukemia cell lines SKM-1, THP-1, and K-562. Treatment with both DFX or DAC promoted apoptosis, induced cell cycle arrest, and inhibited proliferation in all three of these cell lines. The combination of DFX and DAC was much greater than the effect of using either drug alone. DFX showed a synergistic effect with DAC on cell apoptosis in all three cell lines and on cell cycle arrest at the G0/G1 phase in K-562 cells. DFX decreased the ROS levels to varying degrees. In contrast, DAC increased ROS levels and an increase in ROS was also noted when the two drugs were used in combination. Treatment of cells with DAC induced re-expression of ABAT, APAF-1, FADD, HJV, and SMPD3, presumably through demethylation. However the combination of DAC and DFX just had strong synergistic effect on the re-expression of HJV.

Synergistic inhibitory effects of deferasirox in combination with decitabine on leukemia cell lines SKM-1, THP-1, and K-562

PubMed Central

Li, Nianyi; Chen, Qinfen; Gu, Jingwen; Li, Shuang; Zhao, Guangjie; Wang, Wei; Wang, Zhicheng; Wang, Xiaoqin

2017-01-01

A multi-center study from the French Myelodysplastic Syndrome (MDS) Group confirmed that iron chelation therapy is an independent prognostic factor that can increase the survival rate of patients who are suffering from transfusion-dependent low-risk MDS. In this study, we aimed to explore this clinical phenomena in vitro, by exploring the synergistic effect of the iron chelator Deferasirox (DFX) and the DNA methyl transferase inhibitor Decitabine (DAC) in the leukemia cell lines SKM-1, THP-1, and K-562. Treatment with both DFX or DAC promoted apoptosis, induced cell cycle arrest, and inhibited proliferation in all three of these cell lines. The combination of DFX and DAC was much greater than the effect of using either drug alone. DFX showed a synergistic effect with DAC on cell apoptosis in all three cell lines and on cell cycle arrest at the G0/G1 phase in K-562 cells. DFX decreased the ROS levels to varying degrees. In contrast, DAC increased ROS levels and an increase in ROS was also noted when the two drugs were used in combination. Treatment of cells with DAC induced re-expression of ABAT, APAF-1, FADD, HJV, and SMPD3, presumably through demethylation. However the combination of DAC and DFX just had strong synergistic effect on the re-expression of HJV. PMID:28388554

32 CFR 562.7 - Program information.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Program information. 562.7 Section 562.7 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.7 Program information. (a) The Senior ROTC is conducted at military colleges...

32 CFR 562.7 - Program information.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 3 2012-07-01 2009-07-01 true Program information. 562.7 Section 562.7 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS § 562.7 Program information. (a) The Senior ROTC is conducted at military colleges, civilian...

31 CFR 562.101 - Relation of this part to other laws and regulations.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Relation of this part to other laws and regulations. 562.101 Section 562.101 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY IRANIAN HUMAN RIGHTS...

31 CFR 562.101 - Relation of this part to other laws and regulations.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Relation of this part to other laws and regulations. 562.101 Section 562.101 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY IRANIAN HUMAN RIGHTS...

31 CFR 562.101 - Relation of this part to other laws and regulations.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Relation of this part to other laws and regulations. 562.101 Section 562.101 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY IRANIAN HUMAN RIGHTS...

31 CFR 562.101 - Relation of this part to other laws and regulations.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Relation of this part to other laws and regulations. 562.101 Section 562.101 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY IRANIAN HUMAN RIGHTS...

31 CFR 562.309 - United States.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false United States. 562.309 Section 562.309 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... Definitions § 562.309 United States. The term United States means the United States, its territories and...

31 CFR 562.309 - United States.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false United States. 562.309 Section 562.309 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... Definitions § 562.309 United States. The term United States means the United States, its territories and...

31 CFR 562.309 - United States.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false United States. 562.309 Section 562.309 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... Definitions § 562.309 United States. The term United States means the United States, its territories and...

31 CFR 562.309 - United States.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false United States. 562.309 Section 562.309 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF FOREIGN... Definitions § 562.309 United States. The term United States means the United States, its territories and...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at prices offered through the competitive...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance: Treasury 1 2013-07-01 2013-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance MONETARY OFFICES, DEPARTMENT OF THE TREASURY DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance: Treasury 1 2012-07-01 2012-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance MONETARY OFFICES, DEPARTMENT OF THE TREASURY DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance: Treasury 1 2014-07-01 2014-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance MONETARY OFFICES, DEPARTMENT OF THE TREASURY DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at...

31 CFR 56.2 - Sales price.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance: Treasury 1 2011-07-01 2011-07-01 false Sales price. 56.2 Section 56.2 Money and Finance: Treasury Regulations Relating to Money and Finance MONETARY OFFICES, DEPARTMENT OF THE TREASURY DOMESTIC GOLD AND SILVER OPERATIONS SALE OF SILVER § 56.2 Sales price. Sales of silver will be at...

Five novel naphthylisoquinoline alkaloids with growth inhibitory activities against human leukemia cells HL-60, K562 and U937 from stems and leaves of Ancistrocladus tectorius.

PubMed

Jiang, Chao; Li, Zhan-Lin; Gong, Ping; Kang, Sheng-Li; Liu, Ming-Sheng; Pei, Yue-Hu; Jing, Yong-Kui; Hua, Hui-Ming

2013-12-01

Two new 7,6'-coupled naphthylisoquinolines, namely ancistrotectorines A (1) and B (2), two new 5,3'-coupled naphthylisoquinolines, namely ancistrotectorines C (3) and D (4), and one new 7,8-coupled naphthylisoquinoline, namely ancistrotectorine E (5), together with 9 known naphthylisoquinoline alkaloids, hamatine (6), ancistrobertsonine B (7), ancistrocladinine (8), hamatinine (9), ancistrotanzanine A (10), ancistrotanzanine B (11), ancistrotectoriline B (12), 7-epi-ancistrobrevine D (13), and ancistrotectorine (14), were isolated from the 70% EtOH extract of Ancistrocladus tectorius. Their structures were elucidated based on the extensive analysis of spectroscopic data (1D, 2D NMR and MS). Compound 5 exhibited inhibitory activities against HL-60, K562 and U937 cell lines with IC50 values of 1.70, 4.18 and 2.56 μM respectively. © 2013.

DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line.

PubMed

Fares, Mona; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha; Potácová, Zuzana

2015-07-31

We investigated mechanisms of cytotoxicity induced by doxycycline (doxy) and minocycline (mino) in the chronic myeloid leukemia K562 cell line. Doxy and mino induced cell death in exposure-dependent manner. While annexin V/propidium iodide staining was consistent with apoptosis, the morphological changes in Giemsa staining were more equivocal. A pancaspase inhibitor Z-VAD-FMK partially reverted cell death morphology, but concurrently completely prevented PARP cleavage. Mitochondrial involvement was detected as dissipation of mitochondrial membrane potential and cytochrome C release. DNA double strand breaks detected with γH2AX antibody and caspase-2 activation were found early after the treatment start, but caspase-3 activation was a late event. Decrement of Bcl-xL protein levels and electrophoretic shift of Bcl-xL molecule were induced by both drugs. Phosphorylation of Bcl-xL at serine 62 was ruled out. Similarly, Bcr/Abl tyrosine kinase levels were decreased. Lysosomal inhibitor chloroquine restored Bcl-xL and Bcr/Abl protein levels and inhibited caspase-3 activation. Thus, the cytotoxicity of doxy and mino in K562 cells is mediated by DNA damage, Bcl-xL deamidation and lysosomal degradation with activation of mitochondrial pathway of apoptosis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

32 CFR 562.6 - Responsibilities.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 3 2014-07-01 2014-07-01 false Responsibilities. 562.6 Section 562.6 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS... Stovall Street, Alexandria, VA 22332, is the adminstrator of the Department of the Army for ROTC. (b) The...

40 CFR 721.562 - Substituted alkylamine salt.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Substituted alkylamine salt. 721.562 Section 721.562 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.562 Substituted alkylamine salt...

32 CFR 562.4 - Objectives.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 3 2010-07-01 2010-07-01 true Objectives. 562.4 Section 562.4 National Defense Department of Defense (Continued) DEPARTMENT OF THE ARMY ORGANIZED RESERVES RESERVE OFFICERS' TRAINING CORPS... students with potential to serve as commissioned officers in the Regular Army or the US Army Reserve. (b...

Inhibition of DNA-dependent protein kinase catalytic subunit by small molecule inhibitor NU7026 sensitizes human leukemic K562 cells to benzene metabolite-induced apoptosis.

PubMed

You, Hao; Kong, Meng-meng; Wang, Li-ping; Xiao, Xiao; Liao, Han-lin; Bi, Zhuo-yue; Yan, Hong; Wang, Hong; Wang, Chun-hong; Ma, Qiang; Liu, Yan-qun; Bi, Yong-yi

2013-02-01

Benzene is an established leukotoxin and leukemogen in humans. We have previously reported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to mediate the cellular response to DNA double strand break (DSB) caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026), as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phosphorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apoptosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.

Ester of Quinoxaline-7-carboxylate 1,4-di-N-oxide as Apoptosis Inductors in K-562 Cell Line: An in vitro, QSAR and DFT Study.

PubMed

Rivera, Gildardo; Andrade-Ochoa, Sergio; Romero, Manolo S Ortega; Palos, Isidro; Monge, Antonio; Sanchez-Torres, Luvia Enid

2017-01-01

Quinoxalines have shown a wide variety of biological activities including as antitumor agents. The aims of this study were to evaluate the activity of quinoxaline 1,4-di-N-oxide derivatives on K562 cells, the establishment of the mechanism of induced cell death, and the construction of predictive QSAR models. Sixteen esters of quinoxaline-7-carboxylate 1,4-di-N-oxide were evaluated for antitumor activity on K562 chronic myelogenous leukemia cells and their IC50 values were determined. The mechanism of induced cell death by the most active molecule was assessed by flow cytometry and an in silico study was conducted to optimize and calculate theoretical descriptors of all quinoxaline 1,4-di-N-oxide derivatives. QSAR and QPAR models were created using genetic algorithms. Our results show that compounds C5, C7, C10, C12 and C15 had the lowest IC50 of the series. C15 was the most active compound (IC50= 3.02 μg/mL), inducing caspase-dependent apoptotic cell death via the intrinsic pathway. QSAR and QPAR studies are discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

16 CFR 5.62 - Hearing rights of respondent.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Hearing rights of respondent. 5.62 Section 5.62 Commercial Practices FEDERAL TRADE COMMISSION ORGANIZATION, PROCEDURES AND RULES OF PRACTICE STANDARDS OF CONDUCT Disciplinary Actions Concerning Postemployment Conflict of Interest § 5.62 Hearing...

46 CFR 154.562 - Cargo hose: Hydrostatic test.

Code of Federal Regulations, 2010 CFR

2010-10-01

... Hose § 154.562 Cargo hose: Hydrostatic test. Each cargo hose must pass a hydrostatic pressure test at ambient temperature of at least one and a half times its specified maximum working pressure but not more... 46 Shipping 5 2010-10-01 2010-10-01 false Cargo hose: Hydrostatic test. 154.562 Section 154.562...

A nanocomplex of Cu(II) with theophylline drug; synthesis, characterization, and anticancer activity against K562 cell line

NASA Astrophysics Data System (ADS)

Sahlabadi, Maryam; Daryanavard, Marzieh; Hadadzadeh, Hassan; Amirghofran, Zahra

2018-03-01

A new mononuclear of copper (II), [Cu(theophylline)2(H2O)3]·2H2O, has been synthesized by reaction of theophylline (1,3-dimethyl-7H-purine-2,6-dione) with copper (II) nitrate in water. Further, its nanocomplex has been prepared through the three different methods including sonication, grinding, and a combination thereof, sonication-grinding. The prepared nanocomplex was characterized using different techniques including FT-IR, UV-Vis, X-ray diffraction (XRD) analysis, and field-emission scanning electron microscopy (FE-SEM). Moreover, the anticancer activity of the precursor complex, nanocomplex, free theophylline ligand, and the starting copper salt (Cu(NO3)2·3H2O) was investigated against the K562 cell line. The results show that the nanocomplex is an effective nano metal-based anticancer agent with IC50 = 11.7 μM.

31 CFR 562.307 - Property; property interest.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Property; property interest. 562.307 Section 562.307 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE..., or interest or interests therein, present, future, or contingent. ...

31 CFR 562.307 - Property; property interest.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Property; property interest. 562.307 Section 562.307 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE..., or interest or interests therein, present, future, or contingent. ...

31 CFR 562.307 - Property; property interest.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Property; property interest. 562.307 Section 562.307 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE..., or interest or interests therein, present, future, or contingent. ...

31 CFR 562.307 - Property; property interest.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Property; property interest. 562.307 Section 562.307 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE..., or interest or interests therein, present, future, or contingent. ...

33 CFR 183.562 - Metallic fuel lines.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Metallic fuel lines. 183.562...) BOATING SAFETY BOATS AND ASSOCIATED EQUIPMENT Fuel Systems Manufacturer Requirements § 183.562 Metallic fuel lines. (a) Each metallic fuel line that is mounted to the boat structure must be connected to the...

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

31 CFR 562.310 - U.S. financial institution.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false U.S. financial institution. 562.310 Section 562.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... foreign exchange, securities, commodity futures or options, or procuring purchasers and sellers thereof...

31 CFR 562.310 - U.S. financial institution.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false U.S. financial institution. 562.310 Section 562.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... foreign exchange, securities, commodity futures or options, or procuring purchasers and sellers thereof...

31 CFR 562.310 - U.S. financial institution.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false U.S. financial institution. 562.310 Section 562.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... foreign exchange, securities, commodity futures or options, or procuring purchasers and sellers thereof...

31 CFR 562.310 - U.S. financial institution.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false U.S. financial institution. 562.310 Section 562.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... foreign exchange, securities, commodity futures or options, or procuring purchasers and sellers thereof...

The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells.

PubMed

Richardson, D R; Neumannova, V; Nagy, E; Ponka, P

1995-10-15

The iron-responsive element-binding protein (IRE-BP) modulates both ferritin mRNA translation and transferrin receptor (TfR) mRNA stability by binding to specific mRNA sequences called iron-responsive elements (IREs). The regulation of IRE-BP in situ could possibly occur either through its Fe-S cluster and/or via free cysteine sulphydryl groups such as cysteine 437 (Philpott et al, J Biol Chem 268:17655, 1993; and Hirling et al, EMBO J 13:453, 1994). Recently, nitrogen monoxide (NO) has been shown to have markedly different biologic effects depending on its redox state (Lipton et al, Nature 364:626, 1993). Considering this fact, it is conceivable that the NO group, as either the nitrosonium ion (NO+) or nitric oxide (NO+), may regulate IRE-BP activity by S-nitrosylation of key sulphydryl groups or via ligation of NO. to the Fe-S cluster, respectively. This hypothesis has been examined using the NO+ generator, sodium nitroprusside (SNP); the NO. generator, S-nitroso-N-acetylpenicillamine (SNAP); and the NO./peroxynitrite (ONOO-) generator, 3-morpholinosydnonimine hydrochloride (SIN-1). Treatment of K562 cells for 18 hours with SNP (1 mmol/L) resulted in a pronounced decrease in both the RNA-binding activity of IRE-BP and the level of TfR mRNA. In addition, Scatchard analysis showed a marked decrease in the number of specific Tf-binding sites, from 590,000/cell (control) to 170,000/cell (test), and there was also a distinct decrease in Fe uptake. Furthermore, SNP did not decrease cellular viability or proliferation. In contrast, the NO. generator, SNAP (1 mmol/L), increased RNA-binding activity of IRE-BP, the level of TfR mRNA, and the number of TfRs in K562 cells. Moreover, both SNAP (1 mmol/L) and SIN-1 (0.5 mmol/L) reduced cellular proliferation. The results are discussed in context of the possible physiologic role of redox-related species of NO in regulating iron metabolism.

Diterpenes from Xylopia langsdorffiana inhibit cell growth and induce differentiation in human leukemia cells.

PubMed

Castello Branco, Marianna V S; Anazetti, Maristella C; Silva, Marcelo S; Tavares, Josean F; Diniz, Margareth F F Melo; Frungillo, Lucas; Haun, Marcela; Melo, Patrícia S

2009-01-01

Two new diterpenes were isolated from stems and leaves of Xylopia langsdorffiana, ent-atisane-7alpha,16alpha-diol (xylodiol) and ent-7alpha-acetoxytrachyloban-18-oic acid (trachylobane), along with the known 8(17),12E,14-labdatrien-18-oic acid (labdane). We investigated their antitumour effects on HL60, U937 and K562 human leukemia cell lines. We found that xylodiol was the most potent diterpene in inhibiting cell proliferation of HL60, U937 and K562 cells, with mean IC50 values of 90, 80 and 50 microM, respectively. Based on the nitroblue tetrazolium (NBT) reduction assay, all the diterpenes were found to induce terminal differentiation in HL60 and K562 cells, with xylodiol being the most effective. NBT reduction was increased by almost 120% after 12 h exposure of HL60 cells to xylodiol at a concentration lower than the IC50 (50 microM). Thus, xylodiol inhibited human leukemia cell growth in vitro partly by inducing cell differentiation, and merits further studies to examine its mechanism of action as a potential antitumoural agent.

Effect of carbamate pesticides on perforin, granzymes A-B-3/K, and granulysin in human natural killer cells.

PubMed

Li, Qing; Kobayashi, Maiko; Kawada, Tomoyuki

2015-09-01

We previously found that ziram, a carbamate pesticide, significantly reduced perforin, granzyme A (GrA), granzyme B (GrB), granzyme 3/K (Gr3/K), and granulysin (GRN) levels in NK-92MI cells, a human natural killer (NK) cell line. To investigate whether other carbamate pesticides also show similar toxicity on human NK cells, we conducted further experiments with NK-92CI cells, a human NK cell line, using a more sensitive assay. We previously confirmed that NK-92CI cells express CD56, perforin, GrA, GrB, Gr3/K, and GRN and are highly cytotoxic to K562 cells in a chromium release assay, which are more sensitive to organophosphorus pesticides and ziram than the NK-92MI cell line. NK-92CI cells were treated with ziram, thiram, maneb, or carbaryl at various concentrations for 4-24 h at 37°C in vitro. Thereafter, intracellular levels of perforin, GrA, GrB, Gr3/K, and GRN were determined by flow cytometry. It was found that all carbamate pesticides significantly reduced the intracellular levels of perforin, GrA, GrB, Gr3/K, and GRN in NK-92CI cells in a dose-dependent manner. However, the strength of the effect differed among the pesticides, and the order was thiram > ziram > maneb > carbaryl. In addition, it was also found that the degree of the reductions differed among the five proteins, with perforin more sensitive to pesticides than GRN, GrA, GrB, and Gr3/K, and the order was perforin > GRN > Gr3/K ≒ GrA ≒ GrB. © The Author(s) 2015.

40 CFR 56.2 - Scope.

Code of Federal Regulations, 2011 CFR

2011-07-01

... procedures to be employed or policies to be followed by Regional Offices in implementing and enforcing the... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) REGIONAL CONSISTENCY § 56.2 Scope. This part covers actions taken by: (a) Employees in EPA Regional Offices, including Regional...

40 CFR 56.2 - Scope.

Code of Federal Regulations, 2010 CFR

2010-07-01

... procedures to be employed or policies to be followed by Regional Offices in implementing and enforcing the... Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) REGIONAL CONSISTENCY § 56.2 Scope. This part covers actions taken by: (a) Employees in EPA Regional Offices, including Regional...

Downregulation of an Aim-1 Kinase Couples with Megakaryocytic Polyploidization of Human Hematopoietic Cells

PubMed Central

Kawasaki, Akira; Matsumura, Itaru; Miyagawa, Jun-ichiro; Ezoe, Sachiko; Tanaka, Hirokazu; Terada, Yasuhiko; Tatsuka, Masaaki; Machii, Takashi; Miyazaki, Hiroshi; Furukawa, Yusuke; Kanakura, Yuzuru

2001-01-01

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3–dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-rasG12V), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes. PMID:11266445

31 CFR 562.311 - United States person; U.S. person.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false United States person; U.S. person. 562.311 Section 562.311 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued... laws of the United States or any jurisdiction within the United States (including foreign branches), or...

31 CFR 562.311 - United States person; U.S. person.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false United States person; U.S. person. 562.311 Section 562.311 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued... laws of the United States or any jurisdiction within the United States (including foreign branches), or...

Burkholderia cenocepacia K56-2 trimeric autotransporter adhesin BcaA binds TNFR1 and contributes to induce airway inflammation.

PubMed

Mil-Homens, Dalila; Pinto, Sandra N; Matos, Rute G; Arraiano, Cecília; Fialho, Arsenio M

2017-04-01

Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence. © 2016 John Wiley & Sons Ltd.

25 CFR 162.562 - Must a lessee provide insurance for a WSR lease?

Code of Federal Regulations, 2014 CFR

2014-04-01

... 25 Indians 1 2014-04-01 2014-04-01 false Must a lessee provide insurance for a WSR lease? 162.562 Section 162.562 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER LEASES AND PERMITS Wind and Solar Resource Leases Wsr Lease Bonding and Insurance § 162.562 Must a lessee provide...

25 CFR 162.562 - Must a lessee provide insurance for a WSR lease?

Code of Federal Regulations, 2013 CFR

2013-04-01

... 25 Indians 1 2013-04-01 2013-04-01 false Must a lessee provide insurance for a WSR lease? 162.562 Section 162.562 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER LEASES AND PERMITS Wind and Solar Resource Leases Wsr Lease Bonding and Insurance § 162.562 Must a lessee provide...

Anticancer activity of Sargassum oligocystum water extract against human cancer cell lines.

PubMed

Zandi, K; Ahmadzadeh, S; Tajbakhsh, S; Rastian, Z; Yousefi, F; Farshadpour, F; Sartavi, K

2010-08-01

Antitumor drug resistance and side effects of antitumor compounds are the most common problems in medicine. Therefore, finding new antitumor agents with low side effects could be interesting. This study was designed to assay antitumor activity of the extract from brown alga Sargassum oligocystum, gathered from Persian Gulf seashore, against K562 and Daudi human cancer cell lines. The research was performed as an in vitro study. The effect of the alga extract on proliferation of cell lines were measured by two methods: MTT assay and trypan blue exclusion test. The most effective antitumor activity has been shown at concentrations 500 microg/ml and 400 microg/ml of the alga extract against Daudi and K562 cell lines, respectively. The results showed that the extracts of brown alga Sargassum oligocystum have remarkable antitumor activity against K562 and Daudi cell lines. It is justified to be suggested for further research such as algal extract fractionation and purification and in vivo studies in order to formulate natural compounds with antitumor activities.

The 87-kD A gamma-globin enhancer-binding protein is a product of the HOXB2(HOX2H) locus.

PubMed

Sengupta, P K; Lavelle, D E; DeSimone, J

1994-03-01

Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta-globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

12 CFR 562.2 - Regulatory reports.

Code of Federal Regulations, 2010 CFR

2010-01-01

... § 562.2 Regulatory reports. (a) Definition and scope. This section applies to all regulatory reports, as... (TFR) are examples of regulatory reports. Regulatory reports are regulatory documents, not accounting... limited to, the accounting instructions provided in the TFR, guidance contained in OTS regulations...

The combinatorial PP1-binding consensus Motif (R/K)x( (0,1))V/IxFxx(R/K)x(R/K) is a new apoptotic signature.

PubMed

Godet, Angélique N; Guergnon, Julien; Maire, Virginie; Croset, Amélie; Garcia, Alphonse

2010-04-01

Previous studies established that PP1 is a target for Bcl-2 proteins and an important regulator of apoptosis. The two distinct functional PP1 consensus docking motifs, R/Kx((0,1))V/IxF and FxxR/KxR/K, involved in PP1 binding and cell death were previously characterized in the BH1 and BH3 domains of some Bcl-2 proteins. In this study, we demonstrate that DPT-AIF(1), a peptide containing the AIF(562-571) sequence located in a c-terminal domain of AIF, is a new PP1 interacting and cell penetrating molecule. We also showed that DPT-AIF(1) provoked apoptosis in several human cell lines. Furthermore, DPT-APAF(1) a bi-partite cell penetrating peptide containing APAF-1(122-131), a non penetrating sequence from APAF-1 protein, linked to our previously described DPT-sh1 peptide shuttle, is also a PP1-interacting death molecule. Both AIF(562-571) and APAF-1(122-131) sequences contain a common R/Kx((0,1))V/IxFxxR/KxR/K motif, shared by several proteins involved in control of cell survival pathways. This motif combines the two distinct PP1c consensus docking motifs initially identified in some Bcl-2 proteins. Interestingly DPT-AIF(2) and DPT-APAF(2) that carry a F to A mutation within this combinatorial motif, no longer exhibited any PP1c binding or apoptotic effects. Moreover the F to A mutation in DPT-AIF(2) also suppressed cell penetration. These results indicate that the combinatorial PP1c docking motif R/Kx((0,1))V/IxFxxR/KxR/K, deduced from AIF(562-571) and APAF-1(122-131) sequences, is a new PP1c-dependent Apoptotic Signature. This motif is also a new tool for drug design that could be used to characterize potential anti-tumour molecules.

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

PubMed

Verrax, Julien; Vanbever, Stéphanie; Stockis, Julie; Taper, Henryk; Calderon, Pedro Buc

2007-03-15

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells. (c) 2006 Wiley-Liss, Inc.

Potassium Channels Mediate Killing by Human Natural Killer Cells

NASA Astrophysics Data System (ADS)

Schlichter, Lyanne; Sidell, Neil; Hagiwara, Susumu

1986-01-01

Human natural killer (NK) cells in peripheral blood spontaneously recognize and kill a wide variety of target cells. It has been suggested that ion channels are involved in the killing process because there is a Ca-dependent stage and because killing by presensitized cytotoxic T lymphocytes, which in many respects resembles NK killing, is associated with changes in K and Na transport in the target cell. However, no direct evidence exists for ion channels in NK cells or in their target cells. Using the whole-cell variation of the patch-clamp technique, we found a voltage-dependent potassium (K+) current in NK cells. The K+ current was reduced in a dose-dependent manner by the K-channel blockers 4-aminopyridine and quinidine and by the traditional Ca-channel blockers verapamil and Cd2+. We tested the effects of ion-channel blockers on killing of two commonly used target cell lines: K562, which is derived from a human myeloid leukemia, and U937, which is derived from a human histiocytic leukemia. Killing of K562 target cells, determined in a standard 51Cr-release assay, was inhibited in a dose-dependent manner by verapamil, quinidine, Cd2+, and 4-aminopyridine at concentrations comparable to those that blocked the K+ current in NK cells. In K562 target cells only a voltage-dependent Na+ current was found and it was blocked by concentrations of tetrodotoxin that had no effect on killing. Killing of U937 target cells was also inhibited by the two ion-channel blockers tested, quinidine and verapamil. In this cell line only a small K+ current was found that was similar to the one in NK cells. We could not find any evidence of a Ca2+ current in target cells or in NK cells; therefore, our results cannot explain the Ca dependence of killing. Our findings show that there are K channels in NK cells and that these channels play a necessary role in the killing process. In contrast, the endogenous channel type in the target cell is probably not a factor in determining target cell

Dimethyl sulfoxide inactivates the anticancer effect of cisplatin against human myelogenous leukemia cell lines in in vitro assays

PubMed Central

Raghavan, Rahul; Cheriyamundath, Sanith; Madassery, Joseph

2015-01-01

Objectives: To investigate the effect of DMSO on cisplatin induced cytotoxicity (invitro) against K562 (Human mylogenous leukemia) cell line and to study the cisplatin-DMSO adduct formation using UV-spectrophotometer. Materials and methods: Effect of DMSO on the cytotoxicity of cisplatin was studied in K562 (Chronic mylogenous leukemia) cell line by MTT assay. Cisplatin-DMSO adduct formation was studied by continuously monitoring the increase in absorption peaks for 30 minutes using UV-spectrophotometer. Results: 0.1-0.3% DMSO markedly reduced the cytotoxic activity of cisplatin in K562 cells. Cisplatin-DMSO adduct formation was detected using UV-spectrophotometer. Continuous increase in UV absorbance between 250nm-290nm was observed when cisplatin (0.5mg/ml) and DMSO (10%) were mixed. Conclusion: Present study revealed that DMSO inactivates the cytotoxicity of cisplatin. Cisplatin-DMSO mixture showed increased absorbance at 250-290nm. Therefore, using DMSO in invitro assays might result in misinterpretation of actual efficacy of drugs. PMID:26069372

Dimethyl sulfoxide inactivates the anticancer effect of cisplatin against human myelogenous leukemia cell lines in in vitro assays.

PubMed

Raghavan, Rahul; Cheriyamundath, Sanith; Madassery, Joseph

2015-01-01

To investigate the effect of DMSO on cisplatin induced cytotoxicity (invitro) against K562 (Human mylogenous leukemia) cell line and to study the cisplatin-DMSO adduct formation using UV-spectrophotometer. Effect of DMSO on the cytotoxicity of cisplatin was studied in K562 (Chronic mylogenous leukemia) cell line by MTT assay. Cisplatin-DMSO adduct formation was studied by continuously monitoring the increase in absorption peaks for 30 minutes using UV-spectrophotometer. 0.1-0.3% DMSO markedly reduced the cytotoxic activity of cisplatin in K562 cells. Cisplatin-DMSO adduct formation was detected using UV-spectrophotometer. Continuous increase in UV absorbance between 250nm-290nm was observed when cisplatin (0.5mg/ml) and DMSO (10%) were mixed. Present study revealed that DMSO inactivates the cytotoxicity of cisplatin. Cisplatin-DMSO mixture showed increased absorbance at 250-290nm. Therefore, using DMSO in invitro assays might result in misinterpretation of actual efficacy of drugs.

Inhibition of Siah2 Ubiquitin Ligase by Vitamin K3 Attenuates Chronic Myeloid Leukemia Chemo-Resistance in Hypoxic Microenvironment.

PubMed

Huang, Jixian; Lu, Ziyuan; Xiao, Yajuan; He, Bolin; Pan, Chengyun; Zhou, Xuan; Xu, Na; Liu, Xiaoli

2018-02-05

BACKGROUND A hypoxic microenvironment is associated with resistance to tyrosine kinase inhibitors (TKIs) and a poor prognosis in chronic myeloid leukemia (CML). The E3 ubiquitin ligase Siah2 plays a vital role in the regulation of hypoxia response, as well as in leukemogenesis. However, the role of Siah2 in CML resistance is unclear, and it is unknown whether vitaminK3 (a Siah2 inhibitor) can improve the chemo-sensitivity of CML cells in a hypoxic microenvironment. MATERIAL AND METHODS The expression of Siah2 was detected in CML patients (CML-CP and CML-BC), K562 cells, and K562-imatinib-resistant cells (K562-R cells). We measured the expression of PHD3, HIF-1α, and VEGF in both cell lines under normoxia and hypoxic conditions, and the degree of leukemic sensitivity to imatinib and VitaminK3 were evaluated. RESULTS Siah2 was overexpressed in CML-BC patients (n=9) as compared to CML-CP patients (n=13). Similarly, K562-imatinib-resistant cells (K562-R cells) showed a significantly higher expression of Siah2 as compared to K562 cells in a hypoxic microenvironment. Compared to normoxia, under hypoxic conditions, both cell lines had lower PHD3, higher HIF-1α, and higher VEGF expression. Additionally, Vitamin K3 (an inhibitor of Siah2) reversed these changes and promoted a higher degree of leukemic sensitivity to imatinib. CONCLUSIONS Our findings indicate that the Siah2-PHD3- HIF-1α-VEGF axis is an important hypoxic signaling pathway in a leukemic microenvironment. An inhibitor of Siah2, combined with TKIs, might be a promising therapy for relapsing and refractory CML patients.

Evaluation of Synergetic Anticancer Activity of Berberine and Curcumin on Different Models of A549, Hep-G2, MCF-7, Jurkat, and K562 Cell Lines.

PubMed

Balakrishna, Acharya; Kumar, M Hemanth

2015-01-01

Ayurvedic system of medicine is using Berberis aristata and Curcuma longa herbs to treat different diseases including cancer. The study was performed to evaluate the synergetic anticancer activity of Berberine and Curcumin by estimating the inhibition of the cell proliferation by cytotoxicity assay using MTT method on specified human cell lines (A549, Hep-G2, MCF-7, Jurkat, and K562). All the cells were harvested from the culture and seeded in the 96-well assay plates at seeding density of 2.0 × 10(4) cells/well and were incubated for 24 hours. Test items Berberine with Curcumin (1 : 1), Curcumin 95% pure, and Berberine 95% pure were exposed at the concentrations of 1.25, 0.001, and 0.5 mg/mL, respectively, and incubated for a period of 48 hours followed by dispensing MTT solution (5 mg/mL). The cells were incubated at 37 ± 1°C for 4 hours followed by addition of DMSO for dissolving the formazan crystals and absorbance was read at 570 nm. Separate wells were prepared for positive control, controls (only medium with cells), and blank (only medium). The results had proven the synergetic anticancer activity of Berberine with Curcumin inducing cell death greater percentage of >77% when compared to pure curcumin with <54% and pure Berberine with <45% on average on all cell line models.

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

PubMed

Feriotto, G; Calza, R; Bergamini, C M; Griffin, M; Wang, Z; Beninati, S; Ferretti, V; Marzola, E; Guerrini, R; Pagnoni, A; Cavazzini, A; Casciano, F; Mischiati, C

2017-03-01

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Time-series analysis in imatinib-resistant chronic myeloid leukemia K562-cells under different drug treatments.

PubMed

Zhao, Yan-Hong; Zhang, Xue-Fang; Zhao, Yan-Qiu; Bai, Fan; Qin, Fan; Sun, Jing; Dong, Ying

2017-08-01

Chronic myeloid leukemia (CML) is characterized by the accumulation of active BCR-ABL protein. Imatinib is the first-line treatment of CML; however, many patients are resistant to this drug. In this study, we aimed to compare the differences in expression patterns and functions of time-series genes in imatinib-resistant CML cells under different drug treatments. GSE24946 was downloaded from the GEO database, which included 17 samples of K562-r cells with (n=12) or without drug administration (n=5). Three drug treatment groups were considered for this study: arsenic trioxide (ATO), AMN107, and ATO+AMN107. Each group had one sample at each time point (3, 12, 24, and 48 h). Time-series genes with a ratio of standard deviation/average (coefficient of variation) >0.15 were screened, and their expression patterns were revealed based on Short Time-series Expression Miner (STEM). Then, the functional enrichment analysis of time-series genes in each group was performed using DAVID, and the genes enriched in the top ten functional categories were extracted to detect their expression patterns. Different time-series genes were identified in the three groups, and most of them were enriched in the ribosome and oxidative phosphorylation pathways. Time-series genes in the three treatment groups had different expression patterns and functions. Time-series genes in the ATO group (e.g. CCNA2 and DAB2) were significantly associated with cell adhesion, those in the AMN107 group were related to cellular carbohydrate metabolic process, while those in the ATO+AMN107 group (e.g. AP2M1) were significantly related to cell proliferation and antigen processing. In imatinib-resistant CML cells, ATO could influence genes related to cell adhesion, AMN107 might affect genes involved in cellular carbohydrate metabolism, and the combination therapy might regulate genes involved in cell proliferation.

Influence of Hsp70 and HLA-E on the killing of leukemic blasts by cytokine/Hsp70 peptide-activated human natural killer (NK) cells.

PubMed

Stangl, Stefan; Gross, Catharina; Pockley, Alan G; Asea, Alexzander A; Multhoff, Gabriele

2008-01-01

This study compared the effects of the human 70-kDa stress protein (Hsp70) peptide, TKDNNLLGRFELSG (TKD), proinflammatory cytokines, or a combination of both on the repertoire of receptors expressed by human natural killer (NK) cells and their capacity to kill human CX colon carcinoma cells, K562 erythroleukemic cells, and leukemic blasts from two patients with acute myelogenous leukemia. Low-dose interleukin (IL) 2/IL-15 and TKD increase the expression density of activatory (NKG2D, NKp30, NKp44, NKp46, CD94/NKG2C) and inhibitory (CD94/NKG2A) receptors on NK cells. Concomitantly, IL-2/TKD treatment enhances the cytotoxicity of NK cells (as reflected by their secretion of granzyme B) against Hsp70 membrane-positive and human leukocyte antigen (HLA)-E membrane-negative (Hsp70(+)/HLA-E(-)) CX(+) and K562 cells. However, it had no effect on the responsiveness to Hsp70(-)/HLA-E(-) CX(-) cells over that induced by IL-2 alone. The cytotoxicity of IL-2/TKD-activated, purified NK cells and peripheral blood mononuclear cells against Hsp70(+)/HLA-E(+) leukemic blasts was weaker than that against Hsp70(+)/HLA-E(-) K562 cells. Hsp70-blocking and HLA-E transfection experiments confirmed membrane-bound Hsp70 as being a recognition/activatory ligand for NK cells, as cytotoxicity was reduced by the presence of the anti-Hsp70 monoclonal antibody cmHsp70.2 and by inhibiting Hsp70 synthesis using short interference ribonucleic acid. HLA-E was confirmed as an inhibitory ligand, as the extent of NK cell-mediated lysis of K562 cell populations that had been transfected with HLA-E(R) or HLA-E(G) alleles was dependent on the proportion of HLA-E-expressing cells. These findings indicate that Hsp70 (as an activatory molecule) and HLA-E (as an inhibitory ligand) expression influence the susceptibility of leukemic cells to the cytolytic activities of cytokine/TKD-activated NK cells.

Human hematopoietic progenitors express erythropoietin.

PubMed

Stopka, T; Zivny, J H; Stopkova, P; Prchal, J F; Prchal, J T

1998-05-15

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.

Structural Characteristics of the Novel Polysaccharide FVPA1 from Winter Culinary-Medicinal Mushroom, Flammulina velutipes (Agaricomycetes), Capable of Enhancing Natural Killer Cell Activity against K562 Tumor Cells.

PubMed

Jia, Wei; Feng, Jie; Zhang, Jing-Song; Lin, Chi-Chung; Wang, Wen-Han; Chen, Hong-Ge

2017-01-01

FVPA1, a novel polysaccharide, has been isolated from fruiting bodies of the culinary-medicinal mushroom Flammulina velutipes, a historically popular, widely cultivated and consumed functional food with an attractive taste, beneficial nutraceutical properties such as antitumor and immunomodulatory effects, and a number of essential biological activities. The average molecular weight was estimated to be ~1.8 × 104 Da based on high-performance size exclusion chromatography. Sugar analyses, methylation analyses, and 1H, 13C, and 2-dimensional nuclear magnetic resonance spectroscopy revealed the following structure of the repeating units of the FVPA1 polysaccharide Identification of this structure would conceivably lead to better understanding of the nutraceutical functions of this very important edible fungus. Bioactivity tests in vitro indicated that FVPA1 could significantly enhance natural killer cell activity against K562 tumor cells.

49 CFR 236.562 - Minimum rail current required.

Code of Federal Regulations, 2011 CFR

2011-10-01

..., INSPECTION, MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Automatic Train Stop, Train Control and Cab Signal Systems Rules and Instructions; Locomotives § 236.562 Minimum... continuous inductive automatic train stop or train control device to normal condition or to obtain a proceed...

49 CFR 236.562 - Minimum rail current required.

Code of Federal Regulations, 2010 CFR

2010-10-01

..., INSPECTION, MAINTENANCE, AND REPAIR OF SIGNAL AND TRAIN CONTROL SYSTEMS, DEVICES, AND APPLIANCES Automatic Train Stop, Train Control and Cab Signal Systems Rules and Instructions; Locomotives § 236.562 Minimum... continuous inductive automatic train stop or train control device to normal condition or to obtain a proceed...

BVR photometric investigation of galaxy pair KPG 562

NASA Astrophysics Data System (ADS)

Hendy, Y. H. M.

2018-06-01

This work presents BVR photometric observations and analyses for galaxy pair KPG 562 selected from the Karachentsev Catalog of Isolated Pairs of Galaxies. The observations were obtained using the 1.88-m Telescope of the Kottamia Astronomical Observatory (KAO), Egypt. There is no interaction signs assigned for this pair as reported by Karachentsev Catalog. We used the surface photometry technique to obtain photometric parameters for each galaxy of the pair. The isophotal contours, the luminosity profiles, color profiles (B-V, V-R), ellipticity profiles, position angle (PA) profiles and isophotal center-shift (xc, yc) profiles have been presented. The total and absolute magnitude, ellipticity and position angle (PA) were also obtained from the studied galaxy pair. The studied galaxy pair is clearly showing signs of interaction opposed to that found by Karachentsev. We found that the galaxy KPG 562b contains one tidal tail. The length and thickness of tidal tail were obtained and presented in this study.

Myricetin is a novel inhibitor of human inosine 5′-monophosphate dehydrogenase with anti-leukemia activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Huiling; Hu, Qian; Wang, Jingyuan

Human inosine 5′-monophosphate dehydrogenase (hIMPDH) is a rate-limiting enzyme in the de novo biosynthetic pathway of purine nucleotides, playing crucial roles in cellular proliferation, differentiation, and transformation. Dysregulation of hIMPDH expression and activity have been found in a variety of human cancers including leukemia. In this study, we found that myricetin, a naturally occurring phytochemical existed in berries, wine and tea, was a novel inhibitor of human type 1 and type 2 IMPDH (hIMPDH1/2) with IC{sub 50} values of 6.98 ± 0.22 μM and 4.10 ± 0.14 μM, respectively. Enzyme kinetic analysis using Lineweaver-Burk plot revealed that myricetin is a mix-type inhibitor for hIMPDH1/2. Differential scanningmore » fluorimetry and molecular docking simulation data demonstrate that myricetin is capable of binding with hIMPDH1/2. Myricetin treatment exerts potent anti-proliferative and pro-apoptotic effects on K562 human leukemia cells in a dose-dependent manner. Importantly, cytotoxicity of myricetin on K562 cells were markedly attenuated by exogenous addition of guanosine, a salvage pathway of maintaining intracellular pool of guanine nucleotides. Taking together, these results indicate that natural product myricetin exhibits potent anti-leukemia activity by interfering with purine nucleotides biosynthetic pathway through the suppression of hIMPDH1/2 catalytic activity. - Highlights: • Myricetin, a common dietary flavonoid, is a novel inhibitor of hIMPDH1/2. • Myricetin directly binds with hIMPDH1/2 and induces cell cycle arrest and apoptosis of leukemia cells. • The cytotoxicity of myricetin on K562 cells is markedly attenuated by exogenous addition of guanosine.« less

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

PubMed

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-05-06

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification

PubMed Central

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-01-01

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines. PMID:27150638

14 CFR 27.562 - Emergency landing dynamic conditions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Emergency landing dynamic conditions. 27... Conditions § 27.562 Emergency landing dynamic conditions. (a) The rotorcraft, although it may be damaged in... must successfully complete dynamic tests or be demonstrated by rational analysis based on dynamic tests...

14 CFR 23.562 - Emergency landing dynamic conditions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Emergency Landing Conditions § 23.562 Emergency landing dynamic conditions. (a) Each seat/restraint system... dynamic tests conducted in accordance with paragraph (b) of this section: (1) The seat/restraint system... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Emergency landing dynamic conditions. 23...

Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor-modified effector cells.

PubMed

Xiao, Lin; Chen, Can; Li, Zhendong; Zhu, Sumin; Tay, Johan Ck; Zhang, Xi; Zha, Shijun; Zeng, Jieming; Tan, Wee Kiat; Liu, Xin; Chng, Wee Joo; Wang, Shu

2018-03-01

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)-engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

14 CFR 25.562 - Emergency landing dynamic conditions.

Code of Federal Regulations, 2010 CFR

2010-01-01

....562 Emergency landing dynamic conditions. (a) The seat and restraint system in the airplane must be... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Emergency landing dynamic conditions. 25... successfully complete dynamic tests or be demonstrated by rational analysis based on dynamic tests of a similar...

14 CFR 29.562 - Emergency landing dynamic conditions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Emergency landing dynamic conditions. 29... Conditions § 29.562 Emergency landing dynamic conditions. (a) The rotorcraft, although it may be damaged in a... landing must successfully complete dynamic tests or be demonstrated by rational analysis based on dynamic...

7 CFR 56.2 - Designation of official certificates, memoranda, marks, other identifications, and devices for...

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Designation of official certificates, memoranda, marks, other identifications, and devices for purposes of the Agricultural Marketing Act. 56.2 Section 56.2 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing Practices),...

Cytotoxic activity of vitamins K1, K2 and K3 against human oral tumor cell lines.

PubMed

Okayasu, H; Ishihara, M; Satoh, K; Sakagami, H

2001-01-01

Vitamin K1, K2 and K3 were compared for their cytotoxic activity, radical generation and O2- scavenging activity. Among these compounds, vitamin K3 showed the highest cytotoxic activity against human oral tumor cell lines (HSC-2, HSG), human promyelocytic leukemic cell line (HL-60) and human gingival fibroblast (HGF). Vitamin K3 induced internucleosomal DNA fragmentation in HL-60 cells, but not in HSC-2 or HSG cells. The cytotoxic activity of vitamins K2 and K1 was one and two orders lower, respectively, than K3. Vitamin K2, but not vitamin K3, showed tumor-specific cytotoxic action. ESR spectroscopy showed that only vitamin K3 produced radical(s) under alkaline condition and most potently enhanced the radical intensity of sodium ascorbate and scavenged O2- (generated by hypoxanthine-xanthine oxidase reaction system); vitamin K2 was much less active whereas vitamin K1 was inactive. These data suggest that the cytotoxic activity of vitamin K3 is generated by radical-mediated oxidation mechanism and that this vitamin has two opposing actions (that is, antioxidant and prooxidant), depending on the experimental conditions.

Complete nucleotide sequence of the gene for human heparin cofactor II and mapping to chromosomal band 22q11

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herzog, R.; Lutz, S.; Blin, N.

1991-02-05

Heparin cofactor II (HCII) is a 66-kDa plasma glycoprotein that inhibits thrombin rapidly in the presence of dermatan sulfate or heparin. Clones comprising the entire HCII gene were isolated from a human leukocyte genomic library in EMBL-3 {lambda} phage. The sequence of the gene was determined on both strands of DNA (15,849 bp) and included 1,749 bp of 5{prime}-flanking sequence, five exons, four introns, and 476 bp of DNA 3{prime} to the polyadenylation site. Ten complete and one partial Alu repeats were identified in the introns and 5{prime}-flanking region. The HCII gene was regionally mapped on chromosome 22 using rodent-humanmore » somatic cell hybrids, carrying only parts of human chromosome 22, and the chronic myelogenous leukemia cell line K562. With the cDNA probe HCII7.2, containing the entire coding region of the gene, the HCII gene was shown to be amplified 10-20-fold in K562 cells by Southern analysis and in situ hybridization. From these data, the authors concluded that the HCII gene is localized on the chromosomal band 22q11 proximal to the breakpoint cluster region (BCR). Analysis by pulsed-field gel electrophoresis indicated that the amplified HCII gene in K562 cells maps at least 2 Mbp proximal to BCR-1. Furthermore, the HCII7.2 cDNA probe detected two frequent restriction fragment length polymorphisms with the restriction enzymes BamHI and Hind III.« less

40 CFR 60.562-1 - Standards: Process emissions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Compound (VOC) Emissions from the Polymer Manufacturing Industry § 60.562-1 Standards: Process emissions... vent stream from a control device shall have car-sealed opened all valves in the vent system from the emission source to the control device and car-sealed closed all valves in vent system that would lead the...

In Vitro Characterization and Evaluation of the Cytotoxicity Effects of Nisin and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines.

PubMed

Goudarzi, Fariba; Asadi, Asadollah; Afsharpour, Maryam; Jamadi, Robab Hassanvand

2018-05-01

The aim of this study was an in vitro evaluation and comparison of the cytotoxic effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical properties, including formation, size, and shape, were studied using hemolysis, fluorescein isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading efficiency and release kinetics were examined to assess the potential impact of this formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays. Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange (AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter nanoparticles was about 85-90%. The hemolysis test represented their non-cytotoxicities and the FITC images indicated their entrance inside the cells. An increase in the percentage of apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.

Minimally differentiated erythroleukaemia (AML M6 'variant'): a rare subset of AML distinct from AML M6. Groupe Français d'Hématologie Cellulaire.

PubMed

Garand, R; Duchayne, E; Blanchard, D; Robillard, N; Kuhlein, E; Fenneteau, O; Salomon-Nguyen, F; Grange, M J; Rousselot, P; Demur, C

1995-08-01

We describe eight cases of erythroleukaemia distinct from FAB-AML M6, which demonstrate minimal erythroid differentiation not associated with a myeloblastic component. Three infants (including a Down's syndrome) and two adults presented with a de novo leukaemia. One case was preceded by an untreated refractory anaemia with excess of blasts and one by polycythaemia vera. One case presented with an inaugural blast crisis of chronic myeloid leukaemia. In four patients the leukaemic cells showed a proerythroblast-like morphology. The four other were initially classified as undifferentiated AL (two cases) or AML MO (two cases) because of the immature aspect of the cells, their lack of myeloperoxidase activity and the absence of B, T lymphoid and myeloid (My) marker expressions apart from the CD33 antigen. Immunophenotyping in three cases showed an immature erythroblast profile (glycophorins A and B+, spectrin+). In the five others the erythroid nature was recognized by the expression of ABH blood group system on fresh cells (four cases) and glycophorin A on cells after 3 d in vitro culture with erythropoietin (EPO) + IL3 (two cases). Moreover, an erythroid colony growth of leukaemic origin was observed in three patients. In conclusion, the study of erythroid marker expression is of particular importance when immunophenotyping leukaemic cells with a proerythroblast-like morphology or an undifferentiated aspect and a HLA DR-, CD36++, B-, T-, My- (CD33 +/-) phenotype. We propose the term AML M6 'variant' for this rare type of AML.

Closure Report for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Restoration

2012-08-15

This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 562, Waste Systems, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 562 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 as amended). CAU 562 consists of the following 13 Corrective Action Sites (CASs), located in Areas 2, 23, and 25 of the Nevadamore » National Security Site: · CAS 02-26-11, Lead Shot · CAS 02-44-02, Paint Spills and French Drain · CAS 02-59-01, Septic System · CAS 02-60-01, Concrete Drain · CAS 02-60-02, French Drain · CAS 02-60-03, Steam Cleaning Drain · CAS 02-60-04, French Drain · CAS 02-60-05, French Drain · CAS 02-60-06, French Drain · CAS 02-60-07, French Drain · CAS 23-60-01, Mud Trap Drain and Outfall · CAS 23-99-06, Grease Trap · CAS 25-60-04, Building 3123 Outfalls Closure activities began in October 2011 and were completed in April 2012. Activities were conducted according to the Corrective Action Plan for CAU 562 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste and hazardous waste. Some wastes exceeded land disposal limits and required offsite treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite or offsite landfills. NNSA/NSO requests the following: · A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for closure of CAU 562 · The transfer of CAU 562 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO« less

Curcumin induces apoptosis in human leukemic cell lines through an IFIT2-dependent pathway

PubMed Central

Zhang, Yonglu; Kong, Yunyuan; Liu, Shuyuan; Zeng, Lingbing; Wan, Lagen; Zhang, Zhanglin

2017-01-01

ABSTRACT Curcumin, the primary bioactive component isolated from turmeric, has been shown to possess variety of biologic functions including anti-cancer activity. However, molecular mechanisms in different cancer cells are various. In the present study, we demonstrated that curcumin induced G2/M cell cycle arrest and apoptosis by increasing the expression levels of cleaved caspase-3, cleaved PARP and decreasing the expression of BCL−2 in U937 human leukemic cells but not in K562 cells. We found some interferon induced genes, especially interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), were significantly upregulated when treated with curcumin in U937 cells by gene expression chip array, and further confirmed that the expression of IFIT2 was obviously higher in U937 than that in K562 cells by Western blot assay. In addition, inhibiting the expression of IFIT2 by shRNA in U937 rescued curcumin-induced apoptosis and exogenous overexpression of IFIT2 by lentiviral transduction or treating with IFNγ in K562 cells enhanced anti-cancer activity of curcumin. These results indicated for the first time that curcumin induced leukemic cell apoptosis via an IFIT2-dependent signaling pathways. The present study identified a novel mechanism underlying the antitumor effects of curcumin, and may provide a theoretical basis for curcumin combined with interferon in the cancer therapeutics. PMID:28071969

Expansion of NK cells by engineered K562 cells co-expressing 4-1BBL and mMICA, combined with soluble IL-21.

PubMed

Jiang, Bo; Wu, Xuan; Li, Xi-Ning; Yang, Xi; Zhou, Yulai; Yan, Haowei; Wei, An-Hui; Yan, Weiqun

2014-07-01

NK cells hold promise for protecting hosts from cancer and pathogen infection through direct killing and expressing immune-regulatory cytokines. In our study, a genetically modified K562 cell line with surface expression of 4-1BBL and MICA was constructed to expand functional NK cells in vitro for further adoptive immunotherapy against cancer. After a long-term up to 21 day co-culture with newly isolated peripheral blood mononuclear cells (PBMCs) in the presence of soluble IL-21 (sIL-21), notable increase in proportion of expanded NK cells was observed, especially the CD56(bright)CD16(+) subset. Apparent up-regulation of activating receptors CD38, CD69 and NKG2D was detected on expanded NK cells, so did inhibitory receptor CD94; the cytotoxicity of expanded NK cells against target tumor cells exceeded that of NK cells within fresh PBMCs. The intracellular staining showed expanded NK cells produced immune-regulatory IFN-γ. Taken together, we expanded NK cells with significant up-regulation of activating NKG2D and moderate enhancement of cytotoxicity, with IFN-γ producing ability and a more heterogeneous population of NK cells. These findings provide a novel perspective on expanding NK cells in vitro for further biology study and adoptive immunotherapy of NK cells against cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

PubMed

Austruy, E; Bagnis, C; Carbuccia, N; Maroc, C; Birg, F; Dubreuil, P; Mannoni, P; Chabannon, C

1998-01-01

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

40 CFR 180.562 - Flucarbazone-sodium; tolerances for residues.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Flucarbazone-sodium; tolerances for... § 180.562 Flucarbazone-sodium; tolerances for residues. (a) General. Tolerances are established for combined residues of the herbicide flucarbazone-sodium, 4,5-dihydro-3-methoxy-4-methyl-5-oxo-N-[[2...

CIP-36, a novel topoisomerase II-targeting agent, induces the apoptosis of multidrug-resistant cancer cells in vitro.

PubMed

Cao, Bo; Chen, Hong; Gao, Ying; Niu, Cong; Zhang, Yuan; Li, Ling

2015-03-01

The need to overcome cancer multidrug resistance (MDR) has fueled considerable interest in the development of novel synthetic antitumor agents with cytotoxicity against cancer cell lines with MDR. In this study, we aimed to investigate CIP-36, a novel podophyllotoxin derivative, for its inhibitory effects on human cancer cells from multiple sources, particularly cells with MDR in vitro. The human leukemia cell line, K562, and the adriamycin-resistant subline, K562/A02, were exposed to CIP-36 or anticancer agents, and various morphological and biochemical properties were assessed by Hoechst 33342 staining under a fluorescence microscope. Subsequently, cytotoxicity, cell growth curves and the cell cycle were analyzed. Finally, the effects of CIP-36 on topoisomerase IIα (Topo IIα) activity were determined. Treatment with CIP-36 significantly inhibited the growth of the K562 and MDR K562/A02 cells. Our data demonstrated that CIP-36 induced apoptosis, inhibited cell cycle progression and inhibited Topo IIα activity. These findings suggest that CIP-36 has the potential to overcome the multidrug resistance of K562/A02 cells by mediating Topo IIα activity.

Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mizutani, Naoki; College of Life and Health Sciences, Chubu University, Kasugai; Omori, Yukari

2016-02-19

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increasedmore » by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.« less

Encapsulation of Piper cabralanum (Piperaceae) nonpolar extract in poly(methyl methacrylate) by miniemulsion and evaluation of increase in the effectiveness of antileukemic activity in K562 cells.

PubMed

Mendes, Anderson Nogueira; Filgueiras, Lívia Alves; Siqueira, Monica Regina Pimentel; Barbosa, Gleyce Moreno; Holandino, Carla; de Lima Moreira, Davyson; Pinto, José Carlos; Nele, Marcio

2017-01-01

This study aimed to synthesize and characterize nanoparticles (NPs) of poly(methyl methacrylate) (PMMA) and evaluate their ability to incorporate plant extracts with antitumor activity and low dissolution in aqueous media. The extract used was n -hexane partition of the methanol extract of Piper cabralanum (PCA-HEX). PMMA NPs were obtained using the mini-emulsion method, which was able to encapsulate almost 100% of PCA-HEX. The synthesized polymeric particles presented with a size of 200 nm and a negative charge. Cytotoxicity tests by MTT and trypan blue assays showed that NPs without PCA-HEX did not kill leukemic cells (K562 cells). NPs containing PCA-HEX were able to enhance cell death when compared to pure extract. The results showed that PMMA NPs could be useful as a drug delivery system as they can enhance the antitumor activity of the PCA-HEX extract by more than 20-fold. PMMA NPs containing plant extracts with antitumor activities may be an alternative to control the evolution of diseases such as leukemia.

Encapsulation of Piper cabralanum (Piperaceae) nonpolar extract in poly(methyl methacrylate) by miniemulsion and evaluation of increase in the effectiveness of antileukemic activity in K562 cells

PubMed Central

Mendes, Anderson Nogueira; Filgueiras, Lívia Alves; Siqueira, Monica Regina Pimentel; Barbosa, Gleyce Moreno; Holandino, Carla; de Lima Moreira, Davyson; Pinto, José Carlos; Nele, Marcio

2017-01-01

This study aimed to synthesize and characterize nanoparticles (NPs) of poly(methyl methacrylate) (PMMA) and evaluate their ability to incorporate plant extracts with antitumor activity and low dissolution in aqueous media. The extract used was n-hexane partition of the methanol extract of Piper cabralanum (PCA-HEX). PMMA NPs were obtained using the mini-emulsion method, which was able to encapsulate almost 100% of PCA-HEX. The synthesized polymeric particles presented with a size of 200 nm and a negative charge. Cytotoxicity tests by MTT and trypan blue assays showed that NPs without PCA-HEX did not kill leukemic cells (K562 cells). NPs containing PCA-HEX were able to enhance cell death when compared to pure extract. The results showed that PMMA NPs could be useful as a drug delivery system as they can enhance the antitumor activity of the PCA-HEX extract by more than 20-fold. PMMA NPs containing plant extracts with antitumor activities may be an alternative to control the evolution of diseases such as leukemia. PMID:29200848

Ki-67 Expression in Human Tumors Measured by Flow Cytometry

DTIC Science & Technology

1990-01-01

Analyzed Fresh tissues obtained from the lymph node biopsies of 30 patients diagnosed as having non-Hodgkin’s lymphoma, nine biopsies identified as...breast tumors, and eight biopsies identified as colon tumors were included in this study. Cell Lines K562 Cell Line The human ervthroleukemia cell line...petri dish. While holding the tissue with toothed forceps , it wa,-is minced with scissors. Ujsing at transfer pip:., tissue fragments we re aspirated

Eos Negatively Regulates Human γ-globin Gene Transcription during Erythroid Differentiation

PubMed Central

Yu, Hai-Chuan; Zhao, Hua-Lu; Wu, Zhi-Kui; Zhang, Jun-Wu

2011-01-01

Background Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4), a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. Methodology/Principal Findings Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs). DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3) of the β-globin locus control region (LCR), the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C) assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. Conclusions/Significance Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation. PMID:21829552

40 CFR 60.562-2 - Standards: Equipment leaks of VOC.

Code of Federal Regulations, 2010 CFR

2010-07-01

... PROGRAMS (CONTINUED) STANDARDS OF PERFORMANCE FOR NEW STATIONARY SOURCES Standards of Performance for Volatile Organic Compound (VOC) Emissions from the Polymer Manufacturing Industry § 60.562-2 Standards... feature of the pump whereby polymer fluid used to provide lubrication and/or cooling of the pump shaft...

Reengineering cyt b562 for hydrogen production: A facile route to artificial hydrogenases.

PubMed

Sommer, Dayn Joseph; Vaughn, Michael David; Clark, Brett Colby; Tomlin, John; Roy, Anindya; Ghirlanda, Giovanna

2016-05-01

Bioinspired, protein-based molecular catalysts utilizing base metals at the active are emerging as a promising avenue to sustainable hydrogen production. The protein matrix modulates the intrinsic reactivity of organometallic active sites by tuning second-sphere and long-range interactions. Here, we show that swapping Co-Protoporphyrin IX for Fe-Protoporphyrin IX in cytochrome b562 results in an efficient catalyst for photoinduced proton reduction to molecular hydrogen. Further, the activity of wild type Co-cyt b562 can be modulated by a factor of 2.5 by exchanging the coordinating methionine with alanine or aspartic acid. The observed turnover numbers (TON) range between 125 and 305, and correlate well with the redox potential of the Co-cyt b562 mutants. The photosensitized system catalyzes proton reduction with high efficiency even under an aerobic atmosphere, implicating its use for biotechnological applications. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2015 Elsevier B.V. All rights reserved.

Genomic Flexibility of Human Endogenous Retrovirus Type K

PubMed Central

Dube, Derek; Contreras-Galindo, Rafael; He, Shirley; King, Steven R.; Gonzalez-Hernandez, Marta J.; Gitlin, Scott D.; Kaplan, Mark H.

2014-01-01

ABSTRACT Human endogenous retrovirus type K (HERV-K) proviruses are scattered throughout the human genome, but as no infectious HERV-K virus has been detected to date, the mechanism by which these viruses replicated and populated the genome remains unresolved. Here, we provide evidence that, in addition to the RNA genomes that canonical retroviruses package, modern HERV-K viruses can contain reverse-transcribed DNA (RT-DNA) genomes. Indeed, reverse transcription of genomic HERV-K RNA into the DNA form is able to occur in three distinct times and locations: (i) in the virus-producing cell prior to viral release, yielding a DNA-containing extracellular virus particle similar to the spumaviruses; (ii) within the extracellular virus particle itself, transitioning from an RNA-containing particle to a DNA-containing particle; and (iii) after entry of the RNA-containing virus into the target cell, similar to canonical retroviruses, such as murine leukemia virus and HIV. Moreover, using a resuscitated HERV-K virus construct, we show that both viruses with RNA genomes and viruses with DNA genomes are capable of infecting target cells. This high level of genomic flexibility historically could have permitted these viruses to replicate in various host cell environments, potentially assisting in their many integration events and resulting in their high prevalence in the human genome. Moreover, the ability of modern HERV-K viruses to proceed through reverse transcription and package RT-DNA genomes suggests a higher level of replication competency than was previously understood, and it may be relevant in HERV-K-associated human diseases. IMPORTANCE Retroviral elements comprise at least 8% of the human genome. Of all the endogenous retroviruses, HERV-K viruses are the most intact and biologically active. While a modern infectious HERV-K has yet to be found, HERV-K activation has been associated with cancers, autoimmune diseases, and HIV-1 infection. Thus, determining how this

Leaf extracts from Moricandia arvensis promote antiproliferation of human cancer cells, induce apoptosis, and enhance antioxidant activity.

PubMed

Skandrani, Ines; Boubaker, Jihed; Bhouri, Wissem; Limem, Ilef; Kilani, Soumaya; Ben Sghaier, Mohamed; Neffati, Aicha; Bouhlel, Ines; Ghedira, Kamel; Chekir-Ghedira, Leila

2010-01-01

The in vitro antiproliferative, apoptotic, and antioxidant activities from leaf extracts of Moricandia arvensis, which are used in traditional cooking and medicines, were investigated. The MTT assay revealed that only TOF (total oligomer flavonoids), ethyl acetate (EA), chloroform (Chl), and petroleum ether (PE) extracts inhibited the proliferation of K562 cells. Apoptosis plays a very important role in the treatment of cancer by promoting the apoptosis of cancer cells and limiting the concurrent death of normal cells. Thus, the possible effects of M. arvensis extracts on the induction of apoptosis in human leukemic cells (K562 cells) were investigated. The electrophoretic analysis of DNA fragmentation confirms that TOF, Chl, PE, and EA extracts provoke DNA fragmentation. Using the lipid peroxidation inhibitory assay, the antioxidant capacity of M. arvensis extracts was evaluated by the ability of each extract to inhibit malondialdehyde formation. It was revealed that EA and TOF extracts are the most active in scavenging the hydroxyl radicals.

2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate promotes megakaryocytic differentiation of myeloid leukaemia cells and primary human CD34⁺ haematopoietic stem cells.

PubMed

Limb, Jin-Kyung; Song, Doona; Jeon, Mijeong; Han, So-Yeop; Han, Gyoonhee; Jhon, Gil-Ja; Bae, Yun Soo; Kim, Jaesang

2015-04-01

In this study we showed that 2-(trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate [(R)-TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient differentiation of megakaryocytes. Specifically, (R)-TEMOSPho induces cell cycle arrest, cell size increase and polyploidization from K562 and HEL cells, which are used extensively to model megakaryocytic differentiation. In addition, megakaryocyte-specific cell surface markers showed a dramatic increase in expression in response to (R)-TEMOSPho treatment. Importantly, we demonstrated that such megakaryocytic differentiation can also be induced from primary human CD34(+) haematopoietic stem cells. Activation of the PI3K-AKT pathway and, to a lesser extent, the MEK-ERK pathway appears to be required for this process, as blocking with specific inhibitors interferes with the differentiation of K562 cells. A subset of (R)-TEMOSPho-treated K562 cells undergoes spontaneous apoptosis and produces platelets that are apparently functional, as they bind to fibrinogen, express P-selectin and aggregate in response to SFLLRN and AYPGFK, the activating peptides for the PAR1 and PAR4 receptors, respectively. Taken together, these results indicate that (R)-TEMOSPho will be useful for dissecting the molecular mechanisms of megakaryocytic differentiation, and that this class of compounds represents potential therapeutic reagents for thrombocytopenia. Copyright © 2012 John Wiley & Sons, Ltd.

40 CFR 141.562 - My system only has two or fewer filters-is there any special provision regarding individual...

Code of Federal Regulations, 2012 CFR

2012-07-01

... filters-is there any special provision regarding individual filter turbidity monitoring? 141.562 Section... Fewer Than 10,000 People Individual Filter Turbidity Requirements § 141.562 My system only has two or fewer filters—is there any special provision regarding individual filter turbidity monitoring? Yes, if...

40 CFR 141.562 - My system only has two or fewer filters-is there any special provision regarding individual...

Code of Federal Regulations, 2014 CFR

2014-07-01

... filters-is there any special provision regarding individual filter turbidity monitoring? 141.562 Section... Fewer Than 10,000 People Individual Filter Turbidity Requirements § 141.562 My system only has two or fewer filters—is there any special provision regarding individual filter turbidity monitoring? Yes, if...

40 CFR 141.562 - My system only has two or fewer filters-is there any special provision regarding individual...

Code of Federal Regulations, 2010 CFR

2010-07-01

... filters-is there any special provision regarding individual filter turbidity monitoring? 141.562 Section... Fewer Than 10,000 People Individual Filter Turbidity Requirements § 141.562 My system only has two or fewer filters—is there any special provision regarding individual filter turbidity monitoring? Yes, if...

40 CFR 141.562 - My system only has two or fewer filters-is there any special provision regarding individual...

Code of Federal Regulations, 2011 CFR

2011-07-01

... filters-is there any special provision regarding individual filter turbidity monitoring? 141.562 Section... Fewer Than 10,000 People Individual Filter Turbidity Requirements § 141.562 My system only has two or fewer filters—is there any special provision regarding individual filter turbidity monitoring? Yes, if...

40 CFR 141.562 - My system only has two or fewer filters-is there any special provision regarding individual...

Code of Federal Regulations, 2013 CFR

2013-07-01

... filters-is there any special provision regarding individual filter turbidity monitoring? 141.562 Section... Fewer Than 10,000 People Individual Filter Turbidity Requirements § 141.562 My system only has two or fewer filters—is there any special provision regarding individual filter turbidity monitoring? Yes, if...

Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells

PubMed Central

Peng, Xing-Xiang; Tiwari, Amit K.; Wu, Hsiang-Chun; Chen, Zhe-Sheng

2012-01-01

Imatinib, a breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) tyrosine kinase inhibitor (TKI), has revolutionized the treatment of chronic myelogenous leukemia (CML). However, development of multidrug resistance (MDR) limits the use of imatinib. In the present study, we aimed to investigate the mechanisms of cellular resistance to imatinib in CML. Therefore, we established an imatinib-resistant human CML cell line (K562-imatinib) through a stepwise selection process. While characterizing the phenotype of these cells, we found that K562-imatinib cells were 124.6-fold more resistant to imatinib than parental K562 cells. In addition, these cells were cross-resistant to second- and third-generation BCR-ABL TKIs. Western blot analysis and reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that P-glycoprotein (P-gp) and MDR1 mRNA levels were increased in K562-imatinib cells. In addition, accumulation of [14C]6-mercaptopurine (6-MP) was decreased, whereas the ATP-dependent efflux of [14C] 6-MP and [3H]methotrexate transport were increased in K562-imatinib cells. These data suggest that the overexpression of P-gp may play a crucial role in acquired resistance to imatinib in CML K562-imatinib cells. PMID:22098951

Do r/K reproductive strategies apply to human differences?

PubMed

Rushton, J P

1988-01-01

This article discusses the r/K theory of Social Biology and how it relates to humans. The symbols r and K originate in the mathematics of population biology and refer to 2 ends of a continuum in which a compensatory exchange occurs between gamete production (the r-strategy) and longevity (the K-strategy). Both across and within species, r and K strategists differ in a suite of correlated characteristics. Humans are the most K of all. K's supposedly have a longer gestation period, a higher birthweight, a more delayed sexual maturation, a lower sex drive, and a longer life. Studies providing evidence for the expected covariation among K attributes are presented. Additional evidence for r/K theory comes from the comparison of human population known to differ in gamete production. The pattern of racial differences observed to occur in sexual behavior has also been found to exist on numerous other indices of K. For instance, there are racial differences in brain size, intelligence, and maturation rate, among others. The findings suggest that, on the average, Mongoloids are more K than Caucasoids, who in turn, are more K than Negroids. Recently conducted studies have extended the data in favor of r/K theory, and further research is currently underway, including whether r/K attributes underlie individual and social class differences in health and longevity.

Nucleoli in human early erythroblasts (K2, K1, K1/2 cells).

PubMed

Smetana, K; Jirásková, I; Klamová, H

2005-01-01

Human early erythroid precursors classified according to the nuclear size were studied to provide information on nucleoli in these cells using simple cytochemical procedures for demonstration of RNA and proteins of silver-stained nucleolar organizers. K2 cells with nuclear diameter larger than 13 microm and K1 cells with nuclear diameter larger than 9 microm corresponding to proerythroblasts and macroblasts (large basophilic erythroblasts) mostly possessed large irregularly shaped nucleoli with multiple fibrillar centres representing "active nucleoli". K1/2 cells with nuclear diameter smaller than 9 microm corresponding to small basophilic erythroblasts were usually characterized by the presence of micronucleoli representing "inactive nucleolar types". On the other hand, a few K1/2 cells contained large nucleoli with multiple fibrillar centres similar to those present in K2 cells and thus appeared as "microproerythroblasts". The nucleolar asynchrony expressed by the presence of large irregularly shaped nucleoli with multiple nucleoli (active nucleoli) and ring-shaped nucleoli (resting nucleoli) in one and the same nucleus of K2 or K1 cells was not exceptional and might reflect a larger resistance of these cells to negative factors influencing the erythropoiesis. The intranucleolar translocation of silver-stained nucleolus organized regions was noted in K2 cells and might indicate the premature aging of these cells without further differentiation. More studies, however, are required in this direction.

Selective Blockade of Human Natural Killer Cells by a Monoclonal Antibody

NASA Astrophysics Data System (ADS)

Newman, Walter

1982-06-01

A murine monoclonal antibody, 13.1, which blocks human natural killer (NK) cell-mediated lysis, has been developed. Hybridoma 13.1 was derived by fusion of NS-1 cells with spleen cells from mice immunized with an enriched population of NK cells. Supernatants of growing hybridomas were screened for their ability to block NK cell-mediated lysis of K562 targets. Antibody 13.1 is an IgG1 with a single light chain type and it does not fix complement. The 13.1 antigen is expressed on all peripheral blood mononuclear cells, with an antigen density approximately 1/30th that of HLA antigen heavy chain. Pretreatment and washing experiments revealed that inhibition of cytotoxicity occurred at the effector cell level only. Significant blocking was achieved with nanogram quantities of antibody and was not due to toxic effects on NK cells. Likewise, controls with other antibodies of the same subclass demonstrated that blocking was not a consequence of mere binding to NK cells. When a panel of 17 NK cell-susceptible targets was tested, the lysis of only 5 of these was blocked, namely K562, HL-60, KG-1, Daudi, and HEL, a human erythroleukemic cell line. The lysis of 12 human B cell and T cell line targets was not inhibited. In addition to the demonstration that the 13.1 antigen is a crucial cell surface structure involved in NK lysis, a heterogeneity of target cell recognition has been revealed that argues for the proposition that individual NK cells have multiple recognitive capabilities.

Immunohistochemical analysis of S6K1 and S6K2 localization in human breast tumors.

PubMed

Filonenko, Valeriy V; Tytarenko, Ruslana; Azatjan, Sergey K; Savinska, Lilya O; Gaydar, Yuriy A; Gout, Ivan T; Usenko, Vasiliy S; Lyzogubov, Valeriy V

2004-12-01

To perform an immunohistochemical analysis of human breast adenomas and adenocarcinomas as well as normal breast tissues in respect of S6 ribosomal protein kinase (S6K) expression and localization in normal and transformed cells. The expression level and localization of S6K have been detected in formalin fixed, paraffin embedded sections of normal human breast tissues, adenomas and adenocarcinomas with different grade of differentiation. Immunohistochemical detection of S6K1 and S6K2 in normal human breast tissues and breast tumors were performed using specific monoclonal and polyclonal antibodies against S6K1 and S6K2 with following semiquantitative analysis. The increase of S6K content in the cytoplasm of epithelial cells in benign and malignant tumors has been detected. Nuclear accumulation of S6K1 and to a greater extend S6K2 have been found in breast adenocarcinomas. About 80% of breast adenocarcinomas cases revealed S6K2 nuclear staining comparing to normal tissues. In 31% of cases more then 50% of cancer cells had strong nuclear staining. Accumulation of S6K1 in the nucleus of neoplastic cells has been demonstrated in 25% of cases. Nuclear localization of S6K in the epithelial cells in normal breast tissues has not been detected. Immunohistochemical analysis of S6K1 and S6K2 expression in normal human breast tissues, benign and malignant breast tumors clearly indicates that both kinases are overexpressed in breast tumors. Semiquantitative analysis of peculiarities of S6K localization in normal tissues and tumors revealed that nucleoplasmic accumulation of S6K (especially S6K2) is a distinguishing feature of cancer cells.

A Metabolic Biofuel Cell: Conversion of Human Leukocyte Metabolic Activity to Electrical Currents

PubMed Central

2011-01-01

An investigation of the electrochemical activity of human white blood cells (WBC) for biofuel cell (BFC) applications is described. WBCs isolated from whole human blood were suspended in PBS and introduced into the anode compartment of a proton exchange membrane (PEM) fuel cell. The cathode compartment contained a 50 mM potassium ferricyanide solution. Average current densities between 0.9 and 1.6 μA cm-2 and open circuit potentials (Voc) between 83 and 102 mV were obtained, which were both higher than control values. Cyclic voltammetry was used to investigate the electrochemical activity of the activated WBCs in an attempt to elucidate the mechanism of electron transfer between the cells and electrode. Voltammograms were obtained for the WBCs, including peripheral blood mononuclear cells (PBMCs - a lymphocyte-monocyte mixture isolated on a Ficoll gradient), a B lymphoblastoid cell line (BLCL), and two leukemia cell lines, namely K562 and Jurkat. An oxidation peak at about 363 mV vs. SCE for the PMA (phorbol ester) activated primary cells, with a notable absence of a reduction peak was observed. Oxidation peaks were not observed for the BLCL, K562 or Jurkat cell lines. HPLC confirmed the release of serotonin (5-HT) from the PMA activated primary cells. It is believed that serotonin, among other biochemical species released by the activated cells, contributes to the observed BFC currents. PMID:21569243

The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells.

PubMed

Davitt, Katlin; Babcock, Blake D; Fenelus, Maly; Poon, Chi Kong; Sarkar, Abhishek; Trivigno, Vincent; Zolkind, Paul A; Matthew, Sheena M; Grin'kina, Natalia; Orynbayeva, Zulfiya; Shaikh, Mohammad F; Adler, Victor; Michl, Josef; Sarafraz-Yazdi, Ehsan; Pincus, Matthew R; Bowne, Wilbur B

2014-01-01

We have developed the anti-cancer peptide, PNC-27, which is a membrane-active peptide that binds to the HDM-2 protein expressed in the cancer cell membranes of solid tissue tumor cells and induces transmembrane pore formation in cancer, but not in normal cells, resulting in tumor cell necrosis that is independent of p53 activity in these cells. We now extend our study to non-solid tissue tumor cells, in this case, a primitive, possible stem cell human leukemia cell line (K562) that is also p53-homozygously deleted. Our purpose was twofold: to investigate if these cells likewise express HDM-2 in their plasma membranes and to determine if our anti-cancer peptide induces tumor cell necrosis in these non-solid tissue tumor cells in a manner that depends on the interaction between the peptide and membrane-bound HDM-2. The anti-cancer activity and mechanism of PNC-27, which carries a p53 aa12-26-leader sequence connected on its carboxyl terminal end to a trans-membrane-penetrating sequence or membrane residency peptide (MRP), was studied against p53-null K562 leukemia cells. Murine leukocytes were used as a non-cancer cell control. Necrosis was determined by measuring the lactate dehydrogenase (LDH) release and apoptosis was determined by the detection of Caspases 3 and 7. Membrane colocalization of PNC-27 with HDM-2 was analyzed microscopically using fluorescently labeled antibodies against HDM-2 and PNC-27 peptides. We found that K562 cells strongly express HDM-2 protein in their membranes and that PNC-27 co-localizes with this protein in the membranes of these cells. PNC-27, but not the negative control peptide PNC-29, is selectively cytotoxic to K562 cells, inducing nearly 100 percent cell killing with LDH release. In contrast, this peptide had no effect on the lymphocyte control cells. The results suggest that HDM-2 is expressed in the membranes of non-solid tissue tumor cells in addition to the membranes of solid tissue tumor cells. Since K-562 cells appear to be

Synthesis of Nanodiamond-Daunorubicin Conjugates to Overcome Multidrug Chemoresistance in Leukemia

PubMed Central

Man, Han B.; Kim, Hansung; Kim, Ho-Joong; Robinson, Erik; Liu, Wing Kam; Chow, Edward Kai-Hua; Ho, Dean

2013-01-01

Nanodiamonds (NDs) are promising candidates in nanomedicine, demonstrating significant potential as gene/drug delivery platforms for cancer therapy. We have synthesized ND vectors capable of chemotherapeutic loading and delivery with applications towards chemoresistant leukemia. The loading of Daunorubicin (DNR) onto NDs was optimized by adjusting reaction parameters such as acidity and concentration. The resulting conjugate, a novel therapeutic payload for NDs, was characterized extensively for size, surface charge, and loading efficiency. A K562 human myelogenous leukemia cell line, with multidrug resistance conferred by incremental DNR exposure, was used to demonstrate the efficacy enhancement resulting from ND-based delivery. While resistant K562 cells were able to overcome treatment from DNR alone, as compared with non-resistant K562 cells, NDs were able to improve DNR delivery into resistant K562 cells. By overcoming efflux mechanisms present in this resistant leukemia line, ND-enabled therapeutics have demonstrated the potential to improve cancer treatment efficacy, especially towards resistant strains. PMID:23916889

Synthesis and in vitro antiproliferative activity of 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives against human cancer cell lines.

PubMed

Mallesha, Lingappa; Mohana, Kikkeri N; Veeresh, Bantal; Alvala, Ravi; Mallika, Alvala

2012-01-01

A series of new 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives 6a-j were synthesized by a nucleophilic substitution reaction of 2-methyl-3-(2-piperazin-1-ylethyl)-pyrido[1,2-a]pyrimidin-4-one with various sulfonyl chlorides. The compounds were characterized by different spectral studies. All the compounds were evaluated for their antiproliferative effect using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method against four human cancer cell lines (K562, Colo-205, MDA-MB 231, IMR-32) for the time period of 24 h. Among the series, compounds 6d, 6e and 6i showed good activity on all cell lines except K562, whereas the other compounds in the series exhibited moderate activity. Compound 6d could be a potential anticancer agent and therefore deserves further research.

Corrective Action Decision Document for Corrective Action Unit 562: Waste Systems Nevada Test Site, Nevada, Revision 0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mark Krause

2010-08-01

This Corrective Action Decision Document (CADD) presents information supporting the selection of corrective action alternatives (CAAs) leading to the closure of Corrective Action Unit (CAU) 562, Waste Systems, in Areas 2, 23, and 25 of the Nevada Test Site, Nevada. This complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. Corrective Action Unit 562 comprises the following corrective action sites (CASs): • 02-26-11, Lead Shot • 02-44-02, Paint Spills and French Drainmore » • 02-59-01, Septic System • 02-60-01, Concrete Drain • 02-60-02, French Drain • 02-60-03, Steam Cleaning Drain • 02-60-04, French Drain • 02-60-05, French Drain • 02-60-06, French Drain • 02-60-07, French Drain • 23-60-01, Mud Trap Drain and Outfall • 23-99-06, Grease Trap • 25-60-04, Building 3123 Outfalls The purpose of this CADD is to identify and provide the rationale for the recommendation of CAAs for the 13 CASs within CAU 562. Corrective action investigation (CAI) activities were performed from July 27, 2009, through May 12, 2010, as set forth in the CAU 562 Corrective Action Investigation Plan. The purpose of the CAI was to fulfill the following data needs as defined during the data quality objective (DQO) process: • Determine whether COCs are present. • If COCs are present, determine their nature and extent. • Provide sufficient information and data to complete appropriate corrective actions. A data quality assessment (DQA) performed on the CAU 562 data demonstrated the quality and acceptability of the data for use in fulfilling the DQO data needs. Analytes detected during the CAI were evaluated against appropriate final action levels (FALs) to identify the COCs for each CAS. The results of the CAI identified COCs at 10 of the 13 CASs in CAU 562, and thus

Expression of the transcription factor Evi-1 in human erythroleukemia cell lines and in leukemias.

PubMed

Fontenay-Roupie, M; Bouscary, D; Melle, J; Viguié, F; Picard, F; Guesnu, M; Dreyfus, F

1997-02-01

The Evi-1 proto-oncogene is a zinc finger DNA binding protein. Although activation of the Evi-1 gene has been associated with chromosomal rearrangements of the 3q25-q28 region, ectopic expression of Evi-1 could also be observed in acute myelogenous leukemias and myelodysplastic syndromes without cytogenetic abnormalities of the 3q26 locus. In this study, human erythroleukemic cell lines were screened for the expression of Evi-1 mRNA by northern blotting. Evi-1 was expressed in all the erythroid cell lines, whether undifferentiated (K 562, HEL, LAMA 84) or exhibiting spontaneous terminal erythroid differentiation (KU 812, JK-1). Evi-1 mRNA levels were constant or elevated in hemoglobin-synthesizing KU 812 or K 562 cells in response to erythropoietin or hemin treatment, respectively. In human acute myeloblastic leukemias (AML), 11/30 expressed Evi-1 by RT-PCR. Among these cases, 4/6 erythroleukemias without abnormalities of the 3q25-q28 region were found positive. The presence of acidophilic erythroblasts (15-47% of bone marrow cells) accounted for the existence of a terminal erythroid differentiation in all Evi-1-positive AML M6, whereas one negative case was poorly differentiated and referred to as AML M6 variant. These results suggest that Evi-1 mRNA expression can coexist with erythroid differentiation.

Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells.

PubMed

Mizutani, Naoki; Omori, Yukari; Kawamoto, Yoshiyuki; Sobue, Sayaka; Ichihara, Masatoshi; Suzuki, Motoshi; Kyogashima, Mamoru; Nakamura, Mitsuhiro; Tamiya-Koizumi, Keiko; Nozawa, Yoshinori; Murate, Takashi

2016-02-19

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

Paracrine influence of human perivascular cells on the proliferation of adenocarcinoma alveolar epithelial cells.

PubMed

Kim, Eunbi; Na, Sunghun; An, Borim; Yang, Se-Ran; Kim, Woo Jin; Ha, Kwon-Soo; Han, Eun-Taek; Park, Won Sun; Lee, Chang-Min; Lee, Ji Yoon; Lee, Seung-Joon; Hong, Seok-Ho

2017-03-01

Understanding the crosstalk mechanisms between perivascular cells (PVCs) and cancer cells might be beneficial in preventing cancer development and metastasis. In this study, we investigated the paracrine influence of PVCs derived from human umbilical cords on the proliferation of lung adenocarcinoma epithelial cells (A549) and erythroleukemia cells (TF-1α and K562) in vitro using Transwell® co-culture systems. PVCs promoted the proliferation of A549 cells without inducing morphological changes, but had no effect on the proliferation of TF-1α and K562 cells. To identify the factors secreted from PVCs, conditioned media harvested from PVC cultures were analyzed by antibody arrays. We identified a set of cytokines, including persephin (PSPN), a neurotrophic factor, and a key regulator of oral squamous cell carcinoma progression. Supplementation with PSPN significantly increased the proliferation of A549 cells. These results suggested that PVCs produced a differential effect on the proliferation of cancer cells in a cell-type dependent manner. Further, secretome analyses of PVCs and the elucidation of the molecular mechanisms could facilitate the discovery of therapeutic target(s) for lung cancer.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

PubMed Central

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

PubMed Central

Everett, Peter; Clish, Clary B.; Sukhatme, Vikas P.

2010-01-01

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for

7 CFR 56.2 - Designation of official certificates, memoranda, marks, other identifications, and devices for...

Code of Federal Regulations, 2011 CFR

2011-01-01

... AGRICULTURAL MARKETING ACT OF 1946 AND THE EGG PRODUCTS INSPECTION ACT (CONTINUED) VOLUNTARY GRADING OF SHELL EGGS Grading of Shell Eggs Definitions § 56.2 Designation of official certificates, memoranda, marks...

Stress-induced release of HSC70 from human tumors.

PubMed

Barreto, Alfonso; Gonzalez, John Mario; Kabingu, Edith; Asea, Alexzander; Fiorentino, Susana

2003-04-01

In this study, we demonstrate that the pro-inflammatory cytokine interferon-gamma (IFN-gamma) induces the active release of the constitutive form of the 70-kDa heat shock protein (HSC70) from K562 erythroleukemic cells. Treatment of K562 cells with IFN-gamma induced the upregulation of the inducible form of the 70-kDa heat shock protein (HSP70), but not the constitutive form of HSC70 within the cytosol, in a proteasome-dependent manner. In addition, IFN-gamma induced the downregulation of surface-bound HSC70, but did not significantly alter surface-bound HSP70 expression. These findings indicate that HSC70 can be actively released from tumor cells and is indicative of a previously unknown mechanism by which immune modulators stimulate the release of intracellular HSC70. This mechanism may account for the potent chaperokine activity of heat shock proteins recently observed during heat shock protein-based immunotherapy against a variety of cancers.

CCQM-K11.2 determination of glucose in human serum and CCQM-K12.2 determination of creatinine in human serum

NASA Astrophysics Data System (ADS)

Wise, Stephen A.; Phinney, Karen W.; Duewer, David L.; Sniegoski, Lorna T.; Welch, Michael J.; Pritchett, Jeanita; Pabello, Guiomar; Avila Calderon, Marco A.; Balderas, Miryan; Qinde, Liu; Kooi, Lee Tong; Rego, Eliane; Garrido, Bruno; Allegri, Gabriella; de La Cruz, Marcia; Barrabin, Juliana; Monteiro, Tânia; Lee, Hwashim; Kim, Byungjoo; Delatour, Vincent; Peignaux, Maryline; Kawaguchi, Migaku; Bei, Xu; Can, Quan; Nammoonnoy, Jintana; Schild, Katrin; Ohlendorf, Rüdiger; Henrion, Andre; Ceyhan Gören, Ahmet; Yılmaz, Hasibe; Bilsel, Mine; Konopelko, L.; Krylov, A.; Lopushanskaya, E.

2018-01-01

Glucose and creatinine are two of the most frequently measured substances in human blood/serum for assessing the health status of individuals. Because of their clinical significance, CCQM-K11 glucose in human serum and CCQM-K12 creatinine in human serum were the fourth and fifth key comparisons (KCs) performed by the Organic Analysis Working Group (OAWG). These KCs were conducted in parallel and were completed in 2001. The initial subsequent KCs for glucose, CCQM-K11.1, and creatinine, CCQM-K12.1, were completed in 2005. Measurements for the next KCs for these two measurands, CCQM-K11.2 and CCQM-K12.2, were completed in 2013. While designed as subsequent KCs, systematic discordances between the participants' and the anchor institution's results in both comparisons lead the OAWG to request reference results from two experienced laboratories that had participated in the 2001 comparisons. Based on the totality of the available information, the OAWG converted both CCQM-K11.2 and CCQM-K12.2 to 'Track C' KCs where the key comparison reference value is estimated by consensus. These comparisons highlighted that carrying out comparisons for complex chemical measurements and expecting to be able to treat them under the approaches used for formal CIPM subsequent comparisons is not an appropriate strategy. The approach used here is a compromise to gain the best value from the comparison; it is not an approach that will be used in the future. Instead, the OAWG will focus on Track A and Track C comparisons that are treated as stand-alone entities. Participation in CCQM-K11.2 demonstrates a laboratory's capabilities to measure a polar (pKow > 2), low molecular mass (100 g/mol to 500 g/mol) metabolite in human serum at relatively high concentrations (0.1 mg/g to 10 mg/g). Participation in CCQM-K12.2 demonstrates capabilities to measure similar classes of metabolites at relatively low concentrations (1 μg/g to 30 μg/g). The capabilities required for the analysis of complex

In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rowe, Alexander M.; Brundage, Kathleen M.; Center for Immunopathology and Microbial Pathogenesis, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV 26506

2007-06-01

The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesizedmore » that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.« less

A broad survey of cathepsin K immunoreactivity in human neoplasms.

PubMed

Zheng, Gang; Martignoni, Guido; Antonescu, Cristina; Montgomery, Elizabeth; Eberhart, Charles; Netto, George; Taube, Janis; Westra, William; Epstein, Jonathan I; Lotan, Tamara; Maitra, Anirban; Gabrielson, Edward; Torbenson, Michael; Iacobuzio-Donahue, Christine; Demarzo, Angelo; Shih, Ie Ming; Illei, Peter; Wu, T C; Argani, Pedram

2013-02-01

Cathepsin K is consistently and diffusely expressed in alveolar soft part sarcoma (ASPS) and a subset of translocation renal cell carcinomas (RCCs). However, cathepsin K expression in human neoplasms has not been systematically analyzed. We constructed tissue microarrays (TMA) from a wide variety of human neoplasms, and performed cathepsin K immunohistochemistry (IHC). Only 2.7% of 1,140 carcinomas from various sites exhibited cathepsin K labeling, thus suggesting that among carcinomas, cathepsin K labeling is highly specific for translocation RCC. In contrast to carcinomas, cathepsin K labeling was relatively common (54.6%) in the 414 mesenchymal lesions studied, including granular cell tumor, melanoma, and histiocytic lesions, but not paraganglioma, all of which are in the morphologic differential diagnosis of ASPS. Cathepsin K IHC can be helpful in distinguishing ASPS and translocation RCC from some but not all of the lesions in their differential diagnosis.

Bergamot (Citrus bergamia Risso) fruit extracts as γ-globin gene expression inducers: phytochemical and functional perspectives.

PubMed

Guerrini, Alessandra; Lampronti, Ilaria; Bianchi, Nicoletta; Zuccato, Cristina; Breveglieri, Giulia; Salvatori, Francesca; Mancini, Irene; Rossi, Damiano; Potenza, Rocco; Chiavilli, Francesco; Sacchetti, Gianni; Gambari, Roberto; Borgatti, Monica

2009-05-27

Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia.

Transcription factor GATA-1 regulates human HOXB2 gene expression in erythroid cells.

PubMed

Vieille-Grosjean, I; Huber, P

1995-03-03

The human HOXB2 gene is a member of the vertebrate Hox gene family that contains genes coding for specific developmental stage DNA-binding proteins. Remarkably, within the hematopoietic compartment, genes of the HOXB complex are expressed specifically in erythromegakaryocytic cell lines and, for some of them, in hematopoietic progenitors. Here, we report the study of HOXB2 gene transcriptional regulation in hematopoietic cells, an initial step in understanding the lineage-specific expression of the whole HOXB complex in these cells. We have isolated the HOXB2 5'-flanking sequence and have characterized a promoter fragment extending 323 base pairs upstream from the transcriptional start site, which, in transfection experiments, was sufficient to direct the tissue-specific expression of HOXB2 in the erythroid cell line K562. In this fragment, we have identified a potential GATA-binding site that is essential to the promoter activity as demonstrated by point mutation experiments. Gel shift analysis revealed the formation of a specific complex in both erythroleukemic lines K562 and HEL that could be prevented by the addition of a specific antiserum raised against GATA-1 protein. These findings suggest a regulatory hierarchy in which GATA-1 is upstream of the HOXB2 gene in erythroid cells.

Super-Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions.

PubMed

Cao, Fan; Fang, Yiwen; Tan, Hong Kee; Goh, Yufen; Choy, Jocelyn Yeen Hui; Koh, Bryan Thean Howe; Hao Tan, Jiong; Bertin, Nicolas; Ramadass, Aroul; Hunter, Ewan; Green, Jayne; Salter, Matthew; Akoulitchev, Alexandre; Wang, Wilson; Chng, Wee Joo; Tenen, Daniel G; Fullwood, Melissa J

2017-05-19

Stretched histone regions, such as super-enhancers and broad H3K4me3 domains, are associated with maintenance of cell identity and cancer. We connected super-enhancers and broad H3K4me3 domains in the K562 chronic myelogenous leukemia cell line as well as the MCF-7 breast cancer cell line with chromatin interactions. Super-enhancers and broad H3K4me3 domains showed higher association with chromatin interactions than their typical counterparts. Interestingly, we identified a subset of super-enhancers that overlap with broad H3K4me3 domains and show high association with cancer-associated genes including tumor suppressor genes. Besides cell lines, we could observe chromatin interactions by a Chromosome Conformation Capture (3C)-based method, in primary human samples. Several chromatin interactions involving super-enhancers and broad H3K4me3 domains are constitutive and can be found in both cancer and normal samples. Taken together, these results reveal a new layer of complexity in gene regulation by super-enhancers and broad H3K4me3 domains.

Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

PubMed

Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

2012-06-01

The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3.

PubMed Central

Martell, R. L.; Slapak, C. A.; Levy, S. B.

1997-01-01

The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3. Images Figure 3 PMID:9010020

In-vitro antitumor activity evaluation of hyperforin derivatives.

PubMed

Sun, Feng; Liu, Jin-Yun; He, Feng; Liu, Zhong; Wang, Rui; Wang, Dong-Mei; Wang, Yi-Fei; Yang, De-Po

2011-08-01

The derivatives of hyperforin, namely hyperforin acetate (2), 17,18,22,23,27,28,32,33-octahydrohyperforin acetate (3), and N,N-dicyclohexylamine salt of hyperforin (4), have been investigated for their antitumor properties. In-vitro studies demonstrated that 2 and 4 were active against HeLa (human cervical cancer), A375 (human malignant melanoma), HepG2 (human hepatocellular carcinoma), MCF-7 (human breast cancer), A549 (human nonsmall cell lung cancer), K562 (human chronic myeloid leukemia), and K562/ADR (human adriamycin-resistant K562) cell lines with IC(50) values in the range of 3.2-64.1 μM. The energy differences between highest occupied molecular orbital and lowest unoccupied molecular orbital of 2-4 were calculated to be 0.39778, 0.43106, and 0.30900 a.u., respectively, using the Gaussian 03 software package and ab initio method with the HF/6-311 G* basis set. The result indicated that the biological activity of 4 might be the strongest and that of 3 might be the weakest, which was in accordance with their corresponding antiproliferative effects against the tested tumor cell lines. Compound 4 caused cell cycle arrest at G2/M phase in flow cytometry experiment and induced apoptosis by 4',6-diamidino-2-phenylindole staining and Annexin V-FITC/PI (propidium iodide) double-labeled staining in HepG2 cells. The results indicated a potential for N,N-dicyclohexylamine salt of hyperforin as a new antitumor drug.

Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells.

PubMed

Arumuggam, Niroshaathevi; Melong, Nicole; Too, Catherine Kl; Berman, Jason N; Rupasinghe, Hp Vasantha

2017-01-01

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo . We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway.

Phloridzin docosahexaenoate, a novel flavonoid derivative, suppresses growth and induces apoptosis in T-cell acute lymphoblastic leukemia cells

PubMed Central

Arumuggam, Niroshaathevi; Melong, Nicole; Too, Catherine KL; Berman, Jason N; Rupasinghe, HP Vasantha

2017-01-01

The overall clinical outcome in T-cell acute lymphoblastic leukemia (T-ALL) can be improved by minimizing risk for treatment failure using effective pharmacological adjuvants. Phloridzin (PZ), a flavonoid precursor found in apple peels, was acylated with docosahexaenoic acid (DHA) yielding a novel ester known as phloridzin docosahexaenoate (PZ-DHA). Here, we have studied the cytotoxic effects of PZ-DHA on human leukemia cells using in vitro and in vivo models. The inhibitory effects of PZ-DHA were tested on human Jurkat T-ALL cells in comparison to K562 chronic myeloid leukemia (CML) cells and non-malignant murine T-cells. PZ-DHA, not PZ or DHA alone, reduced cell viability and ATP levels, increased intracellular LDH release, and caused extensive morphological alterations in both Jurkat and K562 cells. PZ-DHA also inhibited cell proliferation, and selectively induced apoptosis in Jurkat and K562 cells while sparing normal murine T-cells. The cytotoxic effects of PZ-DHA on Jurkat cells were associated with caspase activation, DNA fragmentation, and selective down-regulation of STAT3 phosphorylation. PZ-DHA significantly inhibited Jurkat cell proliferation in zebrafish larvae; however, the proliferation of K562 cells was not affected in vivo. We propose that PZ-DHA-induced cytotoxic response is selective towards T-ALL in the presence of a tumor-stromal microenvironment. Prospective studies evaluating the combinatorial effects of PZ-DHA with conventional chemotherapy for T-ALL are underway. PMID:29312799

Urea inhibits NaK2Cl cotransport in human erythrocytes.

PubMed Central

Lim, J; Gasson, C; Kaji, D M

1995-01-01

We examined the effect of urea on NaK2Cl cotransport in human erythrocytes. In erythrocytes from nine normal subjects, the addition of 45 mM urea, a concentration commonly encountered in uremic subjects, inhibited NaK2Cl cotransport by 33 +/- 7%. Urea inhibited NaK2Cl cotransport reversibly, and in a concentration-dependent fashion with half-maximal inhibition at 63 +/- 10 mM. Acute cell shrinkage increased, and acute cell swelling decreased NaK2Cl cotransport in human erythrocytes. Okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, increased NaK2Cl cotransport by nearly 80%, suggesting an important role for these phosphatases in the regulation of NaK2Cl cotransport. Urea inhibited bumetanide-sensitive K influx even when protein phosphatases were inhibited with OA, suggesting that urea acted by inhibiting a kinase. In cells subjected to shrinking and OA pretreatment, maneuvers expected to increase the net phosphorylation, urea inhibited cotransport only minimally, suggesting that urea acted by causing a net dephosphorylation of the cotransport protein, or some key regulatory protein. The finding that concentrations of urea found in uremic subjects inhibited NaK2Cl cotransport, a widespread transport pathway with important physiological functions, suggests that urea is not only a marker for accumulation of other uremic toxins, but may be a significant uremic toxin itself. PMID:7593597

Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism.

PubMed

Bonilla-Porras, Angelica R; Jimenez-Del-Rio, Marlene; Velez-Pardo, Carlos

2011-06-10

Secondary therapy-related acute lymphoblastic leukemia might emerge following chemotherapy and/or radiotherapy for primary malignancies. Therefore, other alternatives should be pursued to treat leukemia. It is shown that vitamin K3- or vitamin C- induced apoptosis in leukemia cells by oxidative stress mechanism involving superoxide anion radical and hydrogen peroxide generation, activation of NF-κB, p53, c-Jun, protease caspase-3 activation and mitochondria depolarization leading to nuclei fragmentation. Cell death was more prominent when Jurkat and K562 cells are exposed to VC and VK3 in a ratio 1000:1 (10 mM: 10 μM) or 100:1 (300 μM: 3 μM), respectively. We provide for the first time in vitro evidence supporting a causative role for oxidative stress in VK3- and VC-induced apoptosis in Jurkat and K562 cells in a domino-like mechanism. Altogether these data suggest that VK3 and VC should be useful in the treatment of leukemia.

23Na and 39K NMR studies of ion transport in human erythrocytes.

PubMed

Ogino, T; Shulman, G I; Avison, M J; Gullans, S R; den Hollander, J A; Shulman, R G

1985-02-01

Ion transport in human erythrocytes was studied by 23Na and 39K NMR with an anionic paramagnetic shift reagent, Dy(P3O10)2(7-). The intra- and extracellular 23Na and 39K NMR signals were well separated (over 10 ppm) at 5 mM concentration of the shift reagent. The NMR visibility of the intracellular Na+ and K+ was determined to be 100% in human and duck erythrocytes. The intracellular ion concentrations were 8.1 +/- 0.8 mM Na+ (n = 7) and 110 +/- 12 mM K+ (n = 4) for fresh human erythrocytes. The ouabain-sensitive net Na+ efflux was 1.75 +/- 0.08 mmol/hr per liter of cells at 37 degrees C (n = 3). The gramicidin-induced ion transport in human erythrocytes was also studied by 23Na and 39K NMR or by simultaneous measurements of 23Na NMR and a K+-selective electrode. The time courses of the Na+ and K+ transport induced by the ionophore were biphasic. The initial rapid fluxes were due to an exchange of Na+ for K+, which were found to occur with a 1:1 stoichiometry. The subsequent slow components were the net Na+ and K+ effluxes rate-limited by the Cl- permeability and accompanied by a reduction in cell volume. The Cl- permeability determined from the NMR measurements of these slow fluxes was 3.2 +/- 0.5 X 10(-8) cm/sec at 25 degrees C (n = 4).

23Na and 39K NMR studies of ion transport in human erythrocytes.

PubMed Central

Ogino, T; Shulman, G I; Avison, M J; Gullans, S R; den Hollander, J A; Shulman, R G

1985-01-01

Ion transport in human erythrocytes was studied by 23Na and 39K NMR with an anionic paramagnetic shift reagent, Dy(P3O10)2(7-). The intra- and extracellular 23Na and 39K NMR signals were well separated (over 10 ppm) at 5 mM concentration of the shift reagent. The NMR visibility of the intracellular Na+ and K+ was determined to be 100% in human and duck erythrocytes. The intracellular ion concentrations were 8.1 +/- 0.8 mM Na+ (n = 7) and 110 +/- 12 mM K+ (n = 4) for fresh human erythrocytes. The ouabain-sensitive net Na+ efflux was 1.75 +/- 0.08 mmol/hr per liter of cells at 37 degrees C (n = 3). The gramicidin-induced ion transport in human erythrocytes was also studied by 23Na and 39K NMR or by simultaneous measurements of 23Na NMR and a K+-selective electrode. The time courses of the Na+ and K+ transport induced by the ionophore were biphasic. The initial rapid fluxes were due to an exchange of Na+ for K+, which were found to occur with a 1:1 stoichiometry. The subsequent slow components were the net Na+ and K+ effluxes rate-limited by the Cl- permeability and accompanied by a reduction in cell volume. The Cl- permeability determined from the NMR measurements of these slow fluxes was 3.2 +/- 0.5 X 10(-8) cm/sec at 25 degrees C (n = 4). PMID:2579385

76 FR 7695 - Iranian Human Rights Abuses Sanctions Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-11

... 562.301 Blocked account; blocked property. 562.302 Effective date. 562.303 Entity. 562.304 Interest. 562.305 Licenses; general and specific. 562.306 Person. 562.307 Property; property interest. 562.308... and acquisition of an interest in blocked property. 562.404 Transactions ordinarily incident to a...

Alabama Course of Study: Humanities, K-12. Bulletin 1983, No. 16.

ERIC Educational Resources Information Center

Alabama State Dept. of Education, Montgomery.

A scope and sequence for incorporating humanities into the existing K-12 curriculum contains 8 sections. Following an introduction, the first section outlines characteristics of an effective humanities program. The second and third sections contain teacher and student objectives for a humanities program, minimum requirements, and alternatives for…

Human milk 90K (Mac-2 BP): possible protective effects against acute respiratory infections.

PubMed

Fornarini, B; Iacobelli, S; Tinari, N; Natoli, C; De Martino, M; Sabatino, G

1999-01-01

Eighty-six children fed human milk were followed prospectively from birth to 12 months of age to assess the effect of milk 90K, a secreted glycoprotein with immune-stimulatory properties, on development of acute respiratory infections (ARI). The level of human milk 90K was inversely related to episodes of ARI (r = - 0.34; P = 0.001). The average 90K level in human milk fed to children who did not develop ARI was significantly higher than in milk fed to children in whom infection occurred on multiple occasions (156.6 +/- 144.8 microg/ml versus 70.9 +/- 92.3 microg/ml; P = 0.001). These data suggest that the protective effects of human milk against ARI may be due in part to immune maturation effects by secreted 90K.

The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serafino, A.; Balestrieri, E.; Pierimarchi, P.

2009-03-10

Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derivedmore » non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.« less

Effects of Vitamin K3 and K5 on Daunorubicin-resistant Human T Lymphoblastoid Leukemia Cells.

PubMed

Nakaoka, Eri; Tanaka, Sachiko; Onda, Kenji; Sugiyama, Kentaro; Hirano, Toshihiko

2015-11-01

Anticancer efficacy of vitamin K derivatives on multidrug-resistant cancer cells has been scarcely investigated. The effects of vitamins K3 and K5 on proliferation of human leukemia MOLT-4 cells and on daunorubicin-resistant MOLT-4/DNR cells were estimated by a WST assay. Apoptotic cells were detected by Annexin V and propidium iodide staining, followed by flow cytometry. Vitamins K3 and K5 significantly inhibited proliferation of leukemic cells at 10 and 100 μM (p<0.05), and these effects were almost equally observed in both MOLT-4 and MOLT/DNR drug-resistant cells. Vitamin K3 induced cell apoptosis at 10 and 100 μM in both MOLT-4 and MOLT-4/DNR cells (p<0.05). Vitamin K5 also increased apoptotic cells, while rather inducing necrotic cell death. Vitamins K3 and K5 suppress MOLT-4 and MOLT-4/DNR cell-proliferation partially through induction of apoptosis, and these vitamin derivatives can overcome drug resistance due to P-glycoprotein expression. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Effect of a streptococcal preparation (OK432) on natural killer activity of tumour-associated lymphoid cells in human ovarian carcinoma and on lysis of fresh ovarian tumour cells.

PubMed Central

Colotta, F.; Rambaldi, A.; Colombo, N.; Tabacchi, L.; Introna, M.; Mantovani, A.

1983-01-01

The streptococcal preparation OK432 was studied for its effects on natural killer (NK) activity of peripheral blood lymphocytes (PBL) from normal donors and from ovarian cancer patients, and of tumour-associated lymphocytes (TAL) from peritoneal effusions. OK432 augmented NK activity against the susceptible K562 line and induced killing of the relatively resistant Raji line. Freshly isolated ovarian carcinoma cells were relatively resistant to killing by unstimulated PBL and TAL. OK432 induced significant, though low, levels of cytotoxicity against 51Cr-labelled ovarian carcinoma cells. Augmentation of killing of fresh tumour cells by OK432 was best observed in a 20 h assay and both autologous and allogeneic targets were lysed. PBL were separated on discontinuous Percoll gradients. Unstimulated and OK432-boosted activity were enriched in the lower density fractions where large granular lymphocytes (LGL) and activity against K562 were found. Thus, OK432 augments NK activity of PBL and TAL in human ovarian carcinomas and induces low, but significant, levels of killing of fresh tumour cells. Effector cells involved in killing of fresh ovarian tumours copurify with LGL on discontinuous gradients of Percoll. PMID:6626452

The humanized anti-human AMHRII mAb 3C23K exerts an anti-tumor activity against human ovarian cancer through tumor-associated macrophages.

PubMed

Bougherara, Houcine; Némati, Fariba; Nicolas, André; Massonnet, Gérald; Pugnière, Martine; Ngô, Charlotte; Le Frère-Belda, Marie-Aude; Leary, Alexandra; Alexandre, Jérôme; Meseure, Didier; Barret, Jean-Marc; Navarro-Teulon, Isabelle; Pèlegrin, André; Roman-Roman, Sergio; Prost, Jean-François; Donnadieu, Emmanuel; Decaudin, Didier

2017-11-21

Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient's tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials.

The humanized anti-human AMHRII mAb 3C23K exerts an anti-tumor activity against human ovarian cancer through tumor-associated macrophages

PubMed Central

Bougherara, Houcine; Némati, Fariba; Nicolas, André; Massonnet, Gérald; Pugnière, Martine; Ngô, Charlotte; Le Frère-Belda, Marie-Aude; Leary, Alexandra; Alexandre, Jérôme; Meseure, Didier; Barret, Jean-Marc; Navarro-Teulon, Isabelle; Pèlegrin, André; Roman-Roman, Sergio; Prost, Jean-François; Donnadieu, Emmanuel; Decaudin, Didier

2017-01-01

Müllerian inhibiting substance, also called anti-Müllerian hormone (AMH), inhibits proliferation and induces apoptosis of AMH type II receptor-positive tumor cells, such as human ovarian cancers (OCs). On this basis, a humanized glyco-engineered monoclonal antibody (3C23K) has been developed. The aim of this study was therefore to experimentally confirm the therapeutic potential of 3C23K in human OCs. We first determined by immunofluorescence, immunohistochemistry and cytofluorometry analyses the expression of AMHRII in patient’s tumors and found that a majority (60 to 80% depending on the detection technique) of OCs were positive for this marker. We then provided evidence that the tumor stroma of OC is enriched in tumor-associated macrophages and that these cells are responsible for 3C23K-induced killing of tumor cells through ADCP and ADCC mechanisms. In addition, we showed that 3C23K reduced macrophages induced-T cells immunosuppression. Finally, we evaluated the therapeutic efficacy of 3C23K alone and in combination with a carboplatin-paclitaxel chemotherapy in a panel of OC Patient-Derived Xenografts. In those experiments, we showed that 3C23K significantly increased the proportion and the quality of chemotherapy-based in vivo responses. Altogether, our data support the potential interest of AMHRII targeting in human ovarian cancers and the evaluation of 3C23K in further clinical trials. PMID:29245952

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

PubMed

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

(39)K nuclear magnetic resonance and a mathematical model of K(+) transport in human erythrocytes.

PubMed

Maher, Anthony D; Chapman, Bogdan E; Kuchel, Philip W

2006-04-01

(39)K nuclear magnetic resonance was used to measure the efflux of K(+) from suspensions of human erythrocytes [red blood cells (RBCs)], that occurred in response to the calcium ionophore, A23187 and calcium ions; the latter activate the Gárdos channel. Signals from the intra- and extracellular populations of (39)K(+) were selected on the basis of their longitudinal relaxation times, T (1), by using an inversion- recovery pulse sequence with the mixing time, tau(1), chosen to null one or other of the signals. Changes in RBC volume consequent upon efflux of the ions also changed the T (1) values so a new theory was implemented to obviate a potential artefact in the data analysis. The velocity of the K(+) efflux mediated by the Gárdos channel was 1.19+/-0.40 mmol (L RBC)(-1) min(-1) at 37 degrees C.

Human endogenous retrovirus K and cancer: Innocent bystander or tumorigenic accomplice?

PubMed

Downey, Ronan F; Sullivan, Francis J; Wang-Johanning, Feng; Ambs, Stefan; Giles, Francis J; Glynn, Sharon A

2015-09-15

Harbored as relics of ancient germline infections, human endogenous retroviruses (HERVs) now constitute up to 8% of our genome. A proportion of this sequence has been co-opted for molecular and cellular processes, beneficial to human physiology, such as the fusogenic activity of the envelope protein, a vital component of placentogenesis. However, the discovery of high levels of HERV-K mRNA and protein and even virions in a wide array of cancers has revealed that HERV-K may be playing a more sinister role-a role as an etiological agent in cancer itself. Whether the presence of this retroviral material is simply an epiphenomenon, or an actual causative factor, is a hotly debated topic. This review will summarize the current state of knowledge regarding HERV-K and cancer and attempt to outline the potential mechanisms by which HERV-K could be involved in the onset and promotion of carcinogenesis. © 2014 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

The c-myb proto-oncogene and microRNA-15a comprise an active autoregulatory feedback loop in human hematopoietic cells

PubMed Central

Zhao, Huiwu; Kalota, Anna; Jin, Shenghao

2009-01-01

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment, proliferation, and differentiation. Given its critical functions, c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)–15a. Using a luciferase reporter assay, we found that miR-15a directly binds the 3′-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic, functionality of binding was shown. The mimic decreased c-Myb expression, and blocked the cells in the G1 phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3′-UTR partially rescued the miR-15a induced cell-cycle block. Of interest, the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally, in studies using normal human CD34+ cells, we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation, and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate, these findings suggest the presence of a c-Myb–miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis. PMID:18818396

The k-d Tree: A Hierarchical Model for Human Cognition.

ERIC Educational Resources Information Center

Vandendorpe, Mary M.

This paper discusses a model of information storage and retrieval, the k-d tree (Bentley, 1975), a binary, hierarchical tree with multiple associate terms, which has been explored in computer research, and it is suggested that this model could be useful for describing human cognition. Included are two models of human long-term memory--networks and…

Granzyme M and K release in human experimental endotoxemia.

PubMed

Wensink, Annette C; Wiewel, Maryse A; Jongeneel, Lieneke H; Boes, Marianne; van der Poll, Tom; Hack, C Erik; Bovenschen, Niels

2016-07-01

Granzymes are serine proteases involved in killing of tumor cells and virally infected cells. However, granzymes are also upregulated in blood under inflammatory conditions and contribute to cytokine release and processing. Here, we show that granzyme M (GrM) and to a lesser extent GrK are transiently elevated in the circulation following LPS administration in humans. GrM is released upon stimulation of whole blood with LPS or the gram-negative bacteria Escherichia coli BL21, Pseudomonas aeruginosa, and Neisseria meningitidis. GrK is only released upon stimulation with P. aeruginosa. Thus, GrM and GrK are differentially released in response to LPS and gram-negative bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

p21 as a Transcriptional Co-Repressor of S-Phase and Mitotic Control Genes

PubMed Central

Ferrándiz, Nuria; Caraballo, Juan M.; García-Gutierrez, Lucía; Devgan, Vikram; Rodriguez-Paredes, Manuel; Lafita, M. Carmen; Bretones, Gabriel; Quintanilla, Andrea; Muñoz-Alonso, M. Jose; Blanco, Rosa; Reyes, Jose C.; Agell, Neus; Delgado, M. Dolores; Dotto, G. Paolo; León, Javier

2012-01-01

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes. PMID:22662213

Elements in human serum—CCQM-K139

NASA Astrophysics Data System (ADS)

Shin, Richard; Dewi, Fransiska; Tong, Benny; Wah, Leung Ho; Saxby, David; Armishaw, Paul; Ivanova, Veronika; Feng, Liuxing; Wang, Jun; Estela del Castillo Busto, M.; Fisicaro, Paola; Rienitz, Olaf; Fung, Wai-Hong; Ho-pan Yau, Michael; Yim, Yong-Hyeon; Buzoianu, Mirella; Can, Suleyman Z.; Ari, Betul; Cankur, Oktay; Goenaga Infante, Heidi; Pérez-Zambra, Ramiro; Ferreira, Elizabeth; Long, Stephen

2018-01-01

Elements in human serum serve as important biomarkers and their levels reflect the well-being of an individual. Electrolytes such as sodium (Na) and chloride (Cl) are crucial in maintaining the normal distribution of water, osmotic pressure and electrical neutrality in the body. Trace element such as copper (Cu) plays a part in many oxidation-reduction reactions and metalloenzymes. The majority of selenium (Se) exists as selenoproteins which are cofactors in the glutathione peroxidase activity that protects the body against free radicals. Phosphorus (P) is required for strong bones and teeth. It is also indispensable for growth, maintenance and repair of tissues and cells. The key comparison CCQM-K139: elements in human serum was coordinated by the Health Sciences Authority, Singapore. This comparison aimed to enable participating National Metrology Institutes (NMIs) and Designated Institutes (DIs) to demonstrate their competence in the determination of elements (electrolytes and essential elements) in human serum. The five measurands (Na, Cl, Cu, Se and P) selected for this comparison were not covered in the last two comparisons in the clinical area (CCQM-K14 and CCQM-K107) and offered different analytical challenges. Their concentration levels were within the normal biological range. They were also within the range of existing calibration and measurement capability (CMC) claims in the International Bureau of Weights and Measures' Key Comparison Database (BIPM KCDB). Ten institutes participated in the comparison for Na, eight for Cl, eleven for Cu, six for Se and eight for P. For the analysis of Na, Cu, Se and P, most of the participating institutes employed microwave-assisted digestion and acid digestion (with or without heating) sample dissolution. For the analysis of Cl, in addition to the microwave-assisted digestion and acid digestion, a wider variety of techniques were employed. These included matrix separation, alkaline extraction and coulometric titration

Effects of 1,3,5-triphenyl-4,5-dihydro-1H-pyrazole derivatives on cell-cycle and apoptosis in human acute leukemia cell lines.

PubMed

Santos Bubniak, Lorena Dos; Gaspar, Pâmela Cristina; de Moraes, Ana Carolina Rabello; Bigolin, Alisson; de Souza, Rubia Karine; Buzzi, Fátima Campos; Corrêa, Rogério; Filho, Valdir Cechinel; Bretanha, Lizandra Czermainski; Micke, Gustavo Amadeu; Nunes, Ricardo José; Santos-Silva, Maria Cláudia

2017-05-01

Pyrazoline is an important 5-membered nitrogen heterocycle that has been extensively researched. Ten derivatives were synthesized and tested for antileukemic effects on 2 human acute leukemia cell lines, K562 and Jurkat. The most cytotoxic of these derivatives, compound 21, was chosen for investigation of cytotoxicity mechanisms. The results obtained with selectivity calculations revealed that compound 21 is more selective for acute leukemia (K562 and Jurkat cell lines) than for other tumor cell lines. Moreover, compound 21 was not cytotoxic to normal cell lines, indicating a potential use in clinical tests. Compound 21 caused a significant cell cycle arrest in the S-phase in Jurkat cells and increased the proportion of cells in the sub G0/G1 phase in both cell lines. Cells treated with compound 21 demonstrated morphological changes characteristic of apoptosis in the EB/AO assay, confirmed by externalization of phosphatidylserine by the annexin V - fluorescein isothiocyanate method and by DNA fragmentation. An investigation of cytotoxicity mechanisms suggests the involvement of an intrinsic apoptosis pathway due to mitochondrial damage and an increase in the ratio of mitochondrial Bax/Bcl2. Pyrazoline 21 obeyed Lipinski's "rule of five" for drug-likeness. Based on these preliminary results, the antileukemic activity of compound 21 makes it a potential anticancer agent.

Identification and characterization of metabolites of ASP015K, a novel oral Janus kinase inhibitor, in rats, chimeric mice with humanized liver, and humans.

PubMed

Nakada, Naoyuki; Oda, Kazuo

2015-01-01

1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of (14)C-labelled ASP015K were examined using a liquid chromatography-radiometric detector and mass spectrometer (LC-RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC-MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.

Effects of FR235222, a novel HDAC inhibitor, in proliferation and apoptosis of human leukaemia cell lines: role of annexin A1.

PubMed

Petrella, Antonello; D'Acunto, Cosimo Walter; Rodriquez, Manuela; Festa, Michela; Tosco, Alessandra; Bruno, Ines; Terracciano, Stefania; Taddei, Maurizio; Paloma, Luigi Gomez; Parente, Luca

2008-03-01

FR235222, a novel histone deacetylase inhibitor (HDACi), at 50nM caused accumulation of acetylated histone H4, inhibition of cell proliferation and G1 cycle arrest accompanied by increase of p21 and down-regulation of cyclin E in human promyelocytic leukaemia U937 cells. The compound was also able to increase the protein and mRNA levels of annexin A1 (ANXA1) without effects on apoptosis. Similar effects were observed in human chronic myelogenous leukaemia K562 cells and human T cell leukaemia Jurkat cells. Cycle arrest and ANXA1 expression, without significant effects on apoptosis, were also induced by different HDACi like suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). FR235222 at 0.5 microM stimulated apoptosis of all leukaemia cell lines associated to an increased expression of the full-length (37kDa) protein and the appearance of a 33kDa N-terminal cleavage product in both cytosol and membrane. These results suggest that ANXA1 expression may mediate cycle arrest induced by low doses FR235222, whereas apoptosis induced by high doses FR235222 is associated to ANXA1 processing.

Effectiveness of imatinib mesylate over etoposide in the treatment of sensitive and resistant chronic myeloid leukaemia cells in vitro.

PubMed

Husaini, Roslina; Ahmad, Munirah; Zakaria, Zubaidah

2017-06-01

Chronic myeloid leukaemia (CML) is a form of leukaemia derived from the myeloid cell lineage. Imatinib mesylate, the breakpoint cluster region-abelson murine leukeamia kinase inhibitor, is a specific reagent used in the clinical treatment of CML. The DNA topoisomerase II inhibitor, etoposide, is also employed as a therapeutic, though it is used to a lesser extent. The present study aims to evaluate the effects of CML-targeted therapy, utilising imatinib mesylate and etoposide in the in vitro treatment of parental sensitive and adriamycin-resistant CML in the K562 and K562/ADM cell lines, respectively. Preliminary work involved the screening of multidrug resistant (MDR) gene expression, including MDR1, MRP1 and B-cell lymphoma 2 (BCL-2) at the mRNA levels. The sensitive and resistant CML cell lines expressed the MRP1 gene, though the sensitive K562 cells expressed low, almost undetectable levels of MDR1 and BCL-2 genes relative to the K562/ADM cells. Following treatment with imatinib mesylate or etoposide, the IC50 for imatinib mesylate did not differ between the sensitive and resistant cell lines (0.492±0.024 and 0.378±0.029, respectively), indicating that imatinib mesylate is effective in the treatment of CML regardless of cell chemosensitivity. However, the IC50 for etoposide in sensitive K562 cells was markedly lower than that of K562/ADM cells (50.6±16.5 and 194±8.46 µM, respectively), suggesting that the higher expression levels of MDR1 and/or BCL-2 mRNA in resistant cells may be partially responsible for this effect. This is supported by terminal deoxynucleotidyl transferase dUTP nick-end labeling data, whereby a higher percentage of apoptotic cells were found in the sensitive and resistant K562 cells treated with imatinib mesylate (29.3±0.2 and 31.9±16.7%, respectively), whereas etoposide caused significant apoptosis of sensitive K562 cells (18.3±8.35%) relative to K562/ADM cells (5.17±3.3%). In addition, the MDR genes in K562/ADM cells were knocked

A combination of exosomes carrying TSA derived from HLA-A2-positive human white buffy coat and polyI:C for use as a subcellular antitumor vaccination.

PubMed

Ren, Wei-na; Chang, Chun-kang; Fan, Hua-hua; Guo, Fang; Ren, Ya-na; Yang, Jie; Guo, Juan; Li, Xiao

2011-01-01

To improve its antitumor effect, we used human leukocyte antigen -A2 (HLA-A2)-positive human dendritic cell (DC)-derived DEXs (DC-derived exosomes) to support NY-ESO-1 antigen and polyI:C, with the aim of increasing the proliferation of specific cytotoxic T lymphocytes (CTL) in transgenic mice. Mature dendritic cells derived from peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adults with positive HLA-2A. Using centrifuge and membrane ultrafiltration, EXO (exosomes) were extracted from the supernatant of DCs secretions. Transgenic C57 mice were immunized with human-derived tumor testis antigen NY-ESO-1/EXO, with or without polyI:C. Mice were sacrificed four weeks after immunization, and spleen cells were isolated and tested for function. The experiments included antigen-specific CTL proliferation, as tested by dimerization and antitumor effects for K562 cells as well as melanoma, tested at different ratios of effected cells:target cells (0:1, 10:1, 50:1, and 100:1). Dimerization experiments indicated that the effect of DEX/TSA (tumor specific antigens) + PolyI:C was 2.36 ± 1.10% and the control was 0.38 ± 0.31%, while the effect of DEX/TSA was 1.97 ± 0.63% and the control was 0.36 ± 0.07%. Antitumor effects by DEX/TSA: PolyI:C for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 11.14 ± 1.36%, 14.17 ± 0.62%, 15.71 ± 2.48%, and 24.31 ± 2.91%, respectively, for K562 cells. The antitumor effects for DEX/TSA for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 12.23 ± 2.25%, 13.10 ± 1.57%, 15.27 ± 2.93%, and 19.87 ± 2.72%, respectively, for K562 cells. With ratios of 10:1 and 100:1, the antitumor effects of DEX/TSA + PolyI:C were better than for the DEX/TSA group (P < 0.05). However, higher ratios of effecter cells to target cells increased, and there were no significant improvements in antitumor effect for control cells. Combining PolyI:C with DEX/TSA derived from healthy human blood

Characterization of a novel 132-bp exon of the human maxi-K channel.

PubMed

Korovkina, V P; Fergus, D J; Holdiman, A J; England, S K

2001-07-01

The large-conductance Ca2+-activated voltage-dependent K+ channel (maxi-K channel) induces a significant repolarizing current that buffers cell excitability. This channel can derive its diversity by alternative splicing of its transcript-producing isoforms that differ in their sensitivity to voltage and intracellular Ca2+. We have identified a novel 132-bp exon of the maxi-K channel from human myometrial cells that encodes 44 amino acids within the first intracellular loop of the channel protein. Distribution analysis reveals that this exon is expressed predominantly in human smooth muscle tissues with the highest abundance in the uterus and aorta and resembles the previously reported distribution of the total maxi-K channel transcript. Single-channel K+ current measurements in fibroblasts transfected with the maxi-K channel containing this novel 132-bp exon demonstrate that the presence of this insert attenuates the sensitivity to voltage and intracellular Ca2+. Alternative splicing to introduce this 132-bp exon into the maxi-K channel may elicit another mode to modulate cell excitability.

Novel Hybrid Molecules on the Basis of Steroids and (5Z,9Z)-Tetradeca-5,9-dienoic Acid: Synthesis, Anti-Cancer Studies and Human Topoisomerase I Inhibitory Activity.

PubMed

D'yakonov, Vladimir A; Dzhemileva, Lilya U; Tuktarova, Regina A; Ishmukhametova, Svetlana R; Yunusbaeva, Milyausha M; Ramazanova, Ilfir R; Dzhemilev, Usein M

2017-01-01

Novel steroid derivatives of 5Z,9Z-dienoic acids were prepared by the DCC/DMAP-catalyzed esterification of (5Z,9Z)-tetradeca-5,9-dienoic acid with hydroxy steroids. High cytotoxicity towards the HEK293, Jurkat, K562 cancer cell lines and human topoisomerase I (hTop1) inhibitory activity in vitro were found for the synthesized acids. A probable mechanism of topoisomerase I inhibition was hypothesized on the basis of in silico studies resorting to docking. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Calbindin-D9k (CaBP9k) localization and levels of expression in trophoblast cells from human term placenta.

PubMed

Belkacemi, Louiza; Gariépy, Gilles; Mounier, Catherine; Simoneau, Lucie; Lafond, Julie

2004-01-01

During pregnancy, the calcium (Ca(2+)) transport machinery of the placenta is solely responsible for the nutrient supply to the developing fetus, where active Ca(2+) transport occurs from the mother to the fetus. As part of a larger study to determine the role of Ca(2+) in placental transport in vivo, we questioned whether calbindin-D9k (CaBP9k), which is mainly expressed in duodenum, uterus, and placenta of several mammals, is present in cytotrophoblast cells and syncytiotrophoblasts of human term placenta. We were interested in this protein because of its potential importance in serving as an indicator of Ca(2+) availability and utilization in the placenta. Here, we demonstrated that CaBP9k transcript is present in both cell types, with a lower expression in cytotrophoblast cells as compared to syncytiotrophoblasts. Moreover, we showed by immunochemistry that CaBP9k protein was present in cytotrophoblast and syncytiotrophoblast placental tissue sections as well as in cultured cells. The occurrence of CaBP9k protein in trophoblast cells was further confirmed by Western blot analysis. Thus, these results indicate for the first time that CaBP9k is unequivocally expressed by trophoblast cells from human term placenta.

Cytokine activation induces human memory-like NK cells.

PubMed

Romee, Rizwan; Schneider, Stephanie E; Leong, Jeffrey W; Chase, Julie M; Keppel, Catherine R; Sullivan, Ryan P; Cooper, Megan A; Fehniger, Todd A

2012-12-06

Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

Cell cycle-dependent changes in H3K56ac in human cells

PubMed Central

Stejskal, Stanislav; Stepka, Karel; Tesarova, Lenka; Stejskal, Karel; Matejkova, Martina; Simara, Pavel; Zdrahal, Zbynek; Koutna, Irena

2015-01-01

The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication. PMID:26645646

Pro-arrhythmic effects of low plasma [K+] in human ventricle: An illustrated review.

PubMed

Trenor, Beatriz; Cardona, Karen; Romero, Lucia; Gomez, Juan F; Saiz, Javier; Rajamani, Sridharan; Belardinelli, Luiz; Giles, Wayne

2018-05-01

Potassium levels in the plasma, [K + ] o , are regulated precisely under physiological conditions. However, increases (from approx. 4.5 to 8.0mM) can occur as a consequence of, e.g., endurance exercise, ischemic insult or kidney failure. This hyperkalemic modulation of ventricular electrophysiology has been studied extensively. Hypokalemia is also common. It can occur in response to diuretic therapy, following renal dialysis, or during recovery from endurance exercise. In the human ventricle, clinical hypokalemia (e.g., [K + ] o levels of approx. 3.0mM) can cause marked changes in both the resting potential and the action potential waveform, and these may promote arrhythmias. Here, we provide essential background information concerning the main K + -sensitive ion channel mechanisms that act in concert to produce prominent short-term ventricular electrophysiological changes, and illustrate these by implementing recent mathematical models of the human ventricular action potential. Even small changes (~1mM) in [K + ] o result in significant alterations in two different K + currents, I K1 and HERG. These changes can markedly alter in resting membrane potential and/or action potential waveform in human ventricle. Specifically, a reduction in net outward transmembrane K + currents (repolarization reserve) and an increased substrate input resistance contribute to electrophysiological instability during the plateau of the action potential and may promote pro-arrhythmic early after-depolarizations (EADs). Translational settings where these insights apply include: optimal diuretic therapy, and the interpretation of data from Phase II and III trials for anti-arrhythmic drug candidates. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism.

PubMed

Danielsson, Christian; Brask, Johan; Sköld, Anna-Carin; Genead, Rami; Andersson, Agneta; Andersson, Ulf; Stockling, Kenneth; Pehrson, Rickard; Grinnemo, Karl-Henrik; Salari, Sajjad; Hellmold, Heike; Danielsson, Bengt; Sylvén, Christer; Elinder, Fredrik

2013-01-01

Several drugs blocking the rapidly activating potassium (K(r)) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species. Patch-clamp and quantitative-mRNA measurements of K(r) and slowly activating K (K(s)) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The K(r) channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific K(r)-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed K(r)-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The K(s) channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit. This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific K(r)-channel blocker in human, rat, and rabbit embryos provides evidence of K(r)-channel inhibition as a common mechanism for embryonic malformations and death.

Electrogenic transport and K+ ion channel expression by the human endolymphatic sac epithelium

PubMed Central

Kim, Sung Huhn; Kim, Bo Gyung; Kim, Jin Young; Roh, Kyung Jin; Suh, Michelle J.; Jung, JinSei; Moon, In Seok; Moon, Sung K.; Choi, Jae Young

2015-01-01

The endolymphatic sac (ES) is a cystic organ that is a part of the inner ear and is connected to the cochlea and vestibule. The ES is thought to be involved in inner ear ion homeostasis and fluid volume regulation for the maintenance of hearing and balance function. Many ion channels, transporters, and exchangers have been identified in the ES luminal epithelium, mainly in animal studies, but there has been no functional study investigating ion transport using human ES tissue. We designed the first functional experiments on electrogenic transport in human ES and investigated the contribution of K+ channels in the electrogenic transport, which has been rarely identified, even in animal studies, using electrophysiological/pharmacological and molecular biological methods. As a result, we identified functional and molecular evidence for the essential participation of K+ channels in the electrogenic transport of human ES epithelium. The identified K+ channels involved in the electrogenic transport were KCNN2, KCNJ14, KCNK2, and KCNK6, and the K+ transports via those channels are thought to play an important role in the maintenance of the unique ionic milieu of the inner ear fluid. PMID:26655723

Flow cytometric estimation on cytotoxic activity of leaf extracts from seashore plants in subtropical Japan: isolation, quantification and cytotoxic action of (-)-deoxypodophyllotoxin.

PubMed

Masuda, Toshiya; Oyama, Yasuo; Yonemori, Shigetomo; Takeda, Yoshio; Yamazaki, Yuko; Mizuguchi, Shinichi; Nakata, Mami; Tanaka, Tomochika; Chikahisa, Lumi; Inaba, Yuzuru; Okada, Yoshihiko

2002-06-01

The cytotoxic activity of methanol extracts of leaves collected from 39 seashore plants in Iriomote Island, subtropical Japan was examined on human leukaemia cells (K562 cells) using a flow cytometer with two fluorescent probes, ethidium bromide and annexin V-FITC. Five extracts (10 microg/mL) from Hernandia nymphaeaefolia, Cerbera manghas, Pongamia pinnata, Morus australis var. glabra and Thespesia populnea greatly inhibited the growth of K562 cells. When the concentration was decreased to 1 microg/mL, only one extract from H. nymphaeaefolia still inhibited the cell growth. A cytotoxic compound was isolated from the leaves by bioassay-guided fractionation and was identified as (-)-deoxypodophyllotoxin (DPT). The fresh leaves of H. nymphaeaefolia contained a remarkably high amount of DPT (0.21 +/- 0.07% of fresh leaf weight), being clarified by a quantitative HPLC analysis. DPT at 70-80 pM started to inhibit the growth of K562 cells in an all-or-none fashion and at 100 pM or more it produced complete inhibition in all cases. Therefore, the slope of the dose-response curve was very steep. DPT at 100 pM or more decreased the cell viability to 50%-60% and increased the number of cells undergoing apoptosis (annexin V-positive cells). The results indicate that DPT contributes to the cytotoxic action of the extract from the leaves of H. nymphaeaefolia on K562 cells. Copyright 2002 John Wiley & Sons, Ltd.

Biosynthesis and processing of cathepsin K in cultured human osteoclasts.

PubMed

Rieman, D J; McClung, H A; Dodds, R A; Hwang, S M; Holmes, M W; James, I E; Drake, F H; Gowen, M

2001-03-01

Cathepsin K (cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. However, little is known regarding the synthesis, activation, or turnover of the endogenous enzyme in osteoclasts. In this study, we show that mature cat K protein and enzyme activity are localized within osteoclasts. Pulse-chase experiments revealed that, following the synthesis of pro cat K, intracellular conversion to the mature enzyme occurred in a time-dependent manner. Subsequently, the level of mature enzyme decreased. Little or no cat K was observed in the culture media at any timepoint. Pretreatment of osteoclasts with either chloroquine or monensin resulted in complete inhibition of the processing of newly synthesized cat K. In addition, pro cat K demonstrated susceptibility to treatment with N-glycosidase F, suggesting the presence of high-mannose-containing oligosaccharides. Treatment of osteoclasts with the PI3-kinase inhibitor, Wortmannin (WT), not only prevented the intracellular processing of cat K but also resulted in the secretion of proenzyme into the culture media. Taken together, these results suggest that the biosynthesis, processing, and turnover of cat K in human osteoclasts is constitutive and occurs in a manner similar to that of other known cysteine proteases. Furthermore, cat K is not secreted as a proenzyme, but is processed intracellularly, presumably in lysosomal compartments prior to the release of active enzyme into the resorption lacunae.

EFFECT OF THAI SARAPHI FLOWER EXTRACTS ON WT1 AND BCR/ABL PROTEIN EXPRESSION IN LEUKEMIC CELL LINES.

PubMed

Sangkaruk, Rungkarn; Rungrojsakul, Methee; Tima, Singkome; Anuchapreeda, Songyot

2017-01-01

Saraphi (Mammea siamensis) is a Thai traditional herb. In this study, the cytotoxic effects of crude ethanolic and fractional extracts including hexane, ethyl acetate, and methanol fractions from M. siamensis flowers were investigated in order to determine their effect on WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The flowers of M. siamensis were extracted using ethanol. The ethanol flower extract was further fractionated with hexane, ethyl acetate, and methanol. Cytotoxic effects were measured by the MTT assay. Bcr/Abl and WT1 protein levels after treatments were determined by Western blotting. The total cell number was determined via the typan blue exclusion method. The hexane fraction showed the strongest cytotoxic activity on Molt4 and K562 cells, with IC 50 values of 2.6 and 77.6 μg/ml, respectively. The hexane extract decreased Bcr/Abl protein expression in K562 cells by 74.6% and WT1 protein expressions in Molt4 and K562 cells by 68.4 and 72.1%, respectively. Total cell numbers were decreased by 66.2 and 48.7% in Molt4 and K562 cells, respectively. Mammea E/BB (main active compound) significantly decreased both Bcr/Abl and WTlprotein expressions by 75 and 49.5%, respectively when compared to vehicle control. The hexane fraction from M. siamensis flowers inhibited cell proliferation via the suppression of WT1 expression in Molt4 and K562 cells and Bcr/Abl expression in K562 cells. The active compound may be mammea E/BB. Extracts from M. siamensis flowers show promise as naturally occurring anti-cancer drugs.

Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

NASA Astrophysics Data System (ADS)

Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

2016-01-01

The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks.

PubMed

Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

2016-01-22

The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named "DeepMethyl" to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

Phloretin increases the anti-tumor efficacy of intratumorally delivered heat-shock protein 70 kDa (HSP70) in a murine model of melanoma.

PubMed

Abkin, Sergey V; Ostroumova, Olga S; Komarova, Elena Y; Meshalkina, Darya A; Shevtsov, Maxim A; Margulis, Boris A; Guzhova, Irina V

2016-01-01

Recombinant HSP70 chaperone exerts a profound anticancer effect when administered intratumorally. This action is based on the ability of HSP70 to penetrate tumor cells and extract its endogenous homolog. To enhance the efficacy of HSP70 cycling, we employed phloretin, a flavonoid that enhances the pore-forming activity of the chaperone on artificial membranes. Phloretin increased the efficacy of HSP70 penetration in B16 mouse melanoma cells and K-562 human erythroblasts; this was accompanied with increased transport of the endogenous HSP70 to the plasma membrane. Importantly, treatment with HSP70 combined with phloretin led to the elevation of cell sensitivity to cytotoxic lymphocytes by 16-18 % compared to treatment with the chaperone alone. The incubation of K-562 cells with biotinylated HSP70 and phloretin increased the amount of the chaperone released from cells, suggesting that chaperone cycling could trigger a specific anti-tumor response. We studied the effect of the combination of HSP70 and phloretin using B16 melanoma and a novel method of HSP70-gel application. We found that the addition of phloretin to the gel reduced tumor weight almost fivefold compared with untreated mice, while the life span of the animals extended from 25 to 39 days. The increased survival was corroborated by the activation of innate and adaptive immunity; interestingly, HSP70 was more active in induction of CD8+ cell-mediated toxicity and γIFN production while phloretin contributed largely to the CD56+ cell response. In conclusion, the combination of HSP70 with phloretin could be a novel treatment for efficient immunotherapy of intractable cancers such as skin melanoma.

Experimental model for ELF-EMF exposure: Concern for human health

PubMed Central

D’Angelo, C.; Costantini, E.; Kamal, M.A.; Reale, M.

2014-01-01

Low frequency (LF) electromagnetic fields (EMFs) are abundantly present in modern society and in the last 20 years the interest about the possible effect of extremely low frequency (ELF) EMFs on human health has increased progressively. Epidemiological studies, designed to verify whether EMF exposure may be a potential risk factor for health, have led to controversial results. The possible association between EMFs and an increased incidence of childhood leukemia, brain tumors or neurodegenerative diseases was not fully elucidated. On the other hand, EMFs are widely used, in neurology, psychiatry, rheumatology, orthopedics and dermatology, both in diagnosis and in therapy. In vitro studies may help to evaluate the mechanism by which LF-EMFs affect biological systems. Invitro model of wound healing used keratinocytes (HaCaT), neuroblastoma cell line (SH-SY5Y) as a model for analysis of differentiation, metabolism and functions related to neurodegenerative processes, and monocytic cell line (THP-1) was used as a model for inflammation and cytokines production, while leukemic cell line (K562) was used as a model for hematopoietic differentiation. MCP-1, a chemokine that regulates the migration and infiltration of memory T cells, natural killer (NK), monocytes and epithelial cells, has been demonstrated to be induced and involved in various diseases. Since, varying the parameters of EMFs different effects may be observed, we have studied MCP-1 expression in HaCaT, SH-SY5Y, THP-1 and K562 exposed to a sinusoidal EMF at 50 Hz frequency with a flux density of 1 mT (rms). Our preliminary results showed that EMF-exposure differently modifies the expression of MCP-1 in different cell types. Thus, the MCP-1 expression needs to be better determined, with additional studies, with different parameters and times of exposure to ELF-EMF. PMID:25561888

Serial interactome capture of the human cell nucleus.

PubMed

Conrad, Thomas; Albrecht, Anne-Susann; de Melo Costa, Veronica Rodrigues; Sauer, Sascha; Meierhofer, David; Ørom, Ulf Andersson

2016-04-04

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present 'serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs.

Measurement of Nitrogen Hyperfine Structure on the 53 CM (562 MHz) Butyronitrile Line

NASA Astrophysics Data System (ADS)

Dewberry, Christopher T.; Grubbs, Garry S. Grubbs, II; Raphelt, Andrew; Cooke, Stephen A.

2009-06-01

Recent improvements to our cavity-based Fourier transform radiofrequency spectrometer will be presented. Amongst other improvements use of Miteq amp, model AMF-6F-00100400-10-10P (0.1 GHz to 4 GHz, 65 dB gain minimum, 1 dB noise figure maximum) together with shielding from an improved Faraday cage have significantly helped us in this regard. Electromagnetic fields within our near-spherical cavity have been modeled and results will be presented. We have been able to easily resolve the nitrogen hyperfine structure on the ^aQ_{0,-1} transition 1_{1,0} ← 1_{1,1} located at 562 MHz. This result will be discussed.

Frequency of Human Endogenous Retroviral Sequences (HERV) K113 and K115 in the Polish Population, and Their Effect on HIV Infection

PubMed Central

Zwolińska, Katarzyna; Knysz, Brygida; Gąsiorowski, Jacek; Pazgan-Simon, Monika; Gładysz, Andrzej; Sobczyński, Maciej; Piasecki, Egbert

2013-01-01

Background The human genome contains about 8% of endogenous retroviral sequences originated from germ cell infections by exogenous retroviruses during evolution. Most of those sequences are inactive because of accumulation of mutations but some of them are still capable to be transcribed and translated. The latter are insertionally polymorphic HERV-K113 and HERV-K115. It has been suggested that their presence and expression was connected with several human diseases. It is also believed that they could interfere with the replication cycle of exogenous retroviruses, including HIV. Results Prevalence of endogenous retroviral sequences HERV-K113 and HERV-K115 was determined in the Polish population. The frequencies were found as 11.8% for HERV-K113 and 7.92% for HERV-K115. To verify the hypothesis that the presence of these HERVs sequences could affect susceptibility to HIV infection, comparison of a control group (HIV-negative, not exposed to HIV; n = 303) with HIV-positive patients (n = 470) and exposed but uninfected (EU) individuals (n = 121) was performed. Prevalence of HERV-K113 and HERV-K115 in the EU group was 8.26% and 5.71%, respectively. In the HIV(+) group we detected HERV-K113 sequences in 12.98% of the individuals and HERV-K115 sequences in 7.23% of the individuals. There were no statistically significant differences between groups studied. Conclusion The frequency of HERV-K113 and HERV-K115 sequences in Poland were found to be higher than usually shown for European populations. No relation between presence of the HERVs and HIV infection was detected. PMID:24204983

Corrective Action Investigation Plan for Corrective Action Unit 562: Waste Systems Nevada Test Site, Nevada, Revision 0

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alfred Wickline

2009-04-01

Corrective Action Unit 562 is located in Areas 2, 23, and 25 of the Nevada Test Site, which is approximately 65 miles northwest of Las Vegas, Nevada. Corrective Action Unit 562 is comprised of the 13 corrective action sites (CASs) listed below: • 02-26-11, Lead Shot • 02-44-02, Paint Spills and French Drain • 02-59-01, Septic System • 02-60-01, Concrete Drain • 02-60-02, French Drain • 02-60-03, Steam Cleaning Drain • 02-60-04, French Drain • 02-60-05, French Drain • 02-60-06, French Drain • 02-60-07, French Drain • 23-60-01, Mud Trap Drain and Outfall • 23-99-06, Grease Trap • 25-60-04, Buildingmore » 3123 Outfalls These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives. Additional information will be obtained by conducting a corrective action investigation before evaluating corrective action alternatives and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viable corrective action alternatives that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on December 11, 2008, by representatives of the Nevada Division of Environmental Protection; U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office; Stoller-Navarro Joint Venture; and National Security Technologies, LLC. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 562. Appendix A provides a detailed discussion of the DQO methodology and the DQOs specific to each CAS. The scope of the corrective action investigation for CAU 562 includes the following activities: • Move surface debris and/or materials, as needed, to facilitate

Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes.

PubMed

Chai, Sam; Wan, Xiaoping; Nassal, Drew M; Liu, Haiyan; Moravec, Christine S; Ramirez-Navarro, Angelina; Deschênes, Isabelle

2017-06-01

Two-pore K + (K 2p ) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K 2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K 2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K 2p channels in the heart. Comparing quantitative PCR expression of K 2p channels between human heart tissue and iPSC-CMs revealed K 2p 1.1, K 2p 2.1, K 2p 5.1, and K 2p 17.1 to be higher expressed in cHVT, whereas K 2p 3.1 and K 2p 13.1 were higher in iPSC-CMs. Notably, K 2p 17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K 2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K 2p 2.1, K 2p 3.1, K 2p 6.1, and K 2p 17.1. Here, we report the expression level of 10 human K 2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K 2p 17.1 as significantly reduced in niHF tissues and K 2p 4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K 2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. NEW & NOTEWORTHY Two-pore K + (K 2p ) channels are traditionally regarded as merely background leak channels in myriad

Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets.

PubMed

D'Angelo, D D; Davis, M G; Houser, W A; Eubank, J J; Ritchie, M E; Dorn, G W

1995-09-01

Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest

Identification of NaK-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids.

PubMed

Kelly, R A; O'Hara, D S; Mitch, W E; Smith, T W

1986-09-05

Elevated plasma levels of factors with cardiac glycoside-like activity have been implicated in the response to volume expansion in animals and in the pathogenesis of certain human diseases. We recently described four fractions (IR1, EI1, EI2, EI3) from normal human plasma that inhibit NaK-ATPase, displace ouabain from the enzyme, and exhibit digoxin-like immunoreactivity (Kelly, R. A., O'Hara, D. S., Canessa, M. L., Mitch, W. E., and Smith, T. W. (1985) J. Biol. Chem. 260, 11396-11405). In this report, we identify the active component of these plasma fractions as long-chain nonesterified fatty acids (NEFA) and lysophospholipids. These lipids were present in fractions EI1, EI2, and EI3 in quantities sufficient to account for all of the NaK-ATPase inhibitory activity. The digoxin-like immunoreactivity in fraction IR1 could be attributed to hydrocortisone and other endogenous steroids. To explore the nature of the lipid-NaK-ATPase interactions, we examined the effects of various ATP or sodium concentrations on the NaK-ATPase activity measured in the presence of NEFA. Varying sodium did not affect the inhibition of NaK-ATPase by linoleic acid. At less than 0.15 mM ATP, linoleic acid stimulated NaK-ATPase, but at higher ATP concentrations, the enzyme was progressively inhibited. In summary, NEFA and lysophospholipids, at levels similar to those occurring in human plasma, may account for all of the NaK-ATPase inhibitory activity observed in human plasma fractions. These lipids probably do not directly regulate NaK-ATPase in vivo under normal physiologic conditions, but may alter the sodium pump in disease states characterized by abnormalities in lipid metabolism or plasma protein binding.

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

PubMed

Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

1989-04-01

Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

Spatially correlated phenotyping reveals K5-positive luminal progenitor cells and p63-K5/14-positive stem cell-like cells in human breast epithelium.

PubMed

Boecker, Werner; van Horn, Laura; Stenman, Göran; Stürken, Christine; Schumacher, Udo; Loening, Thomas; Liesenfeld, Lukas; Korsching, Eberhard; Gläser, Doreen; Tiemann, Katharina; Buchwalow, Igor

2018-05-09

Understanding the mechanisms regulating human mammary epithelium requires knowledge of the cellular constituents of this tissue. Different and partially contradictory definitions and concepts describing the cellular hierarchy of mammary epithelium have been proposed, including our studies of keratins K5 and/or K14 as markers of progenitor cells. Furthermore, we and others have suggested that the p53 homolog p63 is a marker of human breast epithelial stem cells. In this investigation, we expand our previous studies by testing whether immunohistochemical staining with monospecific anti-keratin antibodies in combination with an antibody against the stem cell marker p63 might help refine the different morphologic phenotypes in normal breast epithelium. We used in situ multilabel staining for p63, different keratins, the myoepithelial marker smooth muscle actin (SMA), the estrogen receptor (ER), and Ki67 to dissect and quantify the cellular components of 16 normal pre- and postmenopausal human breast epithelial tissue samples at the single-cell level. Importantly, we confirm the existence of K5+ only cells and suggest that they, in contrast to the current view, are key luminal precursor cells from which K8/18+ progeny cells evolve. These cells are further modified by the expression of ER and Ki67. We have also identified a population of p63+K5+ cells that are only found in nipple ducts. Based on our findings, we propose a new concept of the cellular hierarchy of human breast epithelium, including K5 luminal lineage progenitors throughout the ductal-lobular axis and p63+K5+ progenitors confined to the nipple ducts.

Inhibition of the mitochondrial protease, ClpP, as a therapeutic strategy for human acute myeloid leuekmia

PubMed Central

Cole, Alicia; Wang, Zezhou; Coyaud, Etienne; Voisin, Veronique; Gronda, Marcela; Jitkova, Yulia; Mattson, Rachel; Hurren, Rose; Babovic, Sonja; Maclean, Neil; Restall, Ian; Wang, Xiaoming; Jeyaraju, Danny V.; Sukhai, Mahadeo A.; Prabha, Swayam; Bashir, Shaheena; Ramakrishnan, Ashwin; Leung, Elisa; Qia, Yi Hua; Zhang, Nianxian; Combes, Kevin R.; Ketela, Troy; Lin, Fengshu; Houry, Walid A.; Aman, Ahmed; Al-awar, Rima; Zheng, Wei; Wienholds, Erno; Xu, Chang Jiang; Dick, John; Wang, Jean C.Y.; Moffat, Jason; Minden, Mark D.; Eaves, Connie J.; Bader, Gary D.; Hao, Zhenyue; Kornblau, Steven M.; Raught, Brian; Schimmer, Aaron D.

2015-01-01

Summary From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in the leukemic cells from approximately half of patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression, but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism. PMID:26058080

Synthesis and anti-cancer activity evaluation of novel prenylated and geranylated chalcone natural products and their analogs.

PubMed

Wang, Hao-Meng; Zhang, Li; Liu, Jiang; Yang, Zhao-Liang; Zhao, Hong-Ye; Yang, Yao; Shen, Di; Lu, Kui; Fan, Zhen-Chuan; Yao, Qing-Wei; Zhang, Yong-Min; Teng, Yu-Ou; Peng, Yu

2015-03-06

Four natural chalcones bearing prenyl or geranyl groups, i.e., bavachalcone (1a), xanthoangelol (1b), isobavachalcone (1c), and isoxanthoangelol (1d) were synthesized by using a regio-selective iodination and the Suzuki coupling reaction as key steps. The first total synthesis of isoxanthoangelol (1d) was achieved in 36% overall yield. A series of diprenylated and digeranylated chalcone analogs were also synthesized by alkylation, regio-selective iodination, aldol condensation, Suzuki coupling and [1,3]-sigmatropic rearrangement. The structures of the 11 new derivatives were confirmed by (1)H NMR, (13)C NMR and HRMS. The anticancer activity of these new chalcone derivatives against human tumor cell line K562 were evaluated by MTT assay in vitro. SAR studies suggested that the 5'-prenylation/geranylation of the chalcones significantly enhance their cytotoxic activity. Among them, Bavachalcone (1a) displayed the most potent cytotoxic activity against K562 with IC50 value of 2.7 μM. The morphology changes and annexin-V/PI staining studies suggested that those chalcone derivatives inhibited the proliferation of K562 cells by inducing apoptosis. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

A recombinant dromedary antibody fragment (VHH or nanobody) directed against human Duffy antigen receptor for chemokines.

PubMed

Smolarek, Dorota; Hattab, Claude; Hassanzadeh-Ghassabeh, Gholamreza; Cochet, Sylvie; Gutiérrez, Carlos; de Brevern, Alexandre G; Udomsangpetch, Rachanee; Picot, Julien; Grodecka, Magdalena; Wasniowska, Kazimiera; Muyldermans, Serge; Colin, Yves; Le Van Kim, Caroline; Czerwinski, Marcin; Bertrand, Olivier

2010-10-01

Fy blood group antigens are carried by the Duffy antigen receptor for chemokines (DARC), a red cells receptor for Plasmodium vivax broadly implicated in human health and diseases. Recombinant VHHs, or nanobodies, the smallest intact antigen binding fragment derivative from the heavy chain-only antibodies present in camelids, were prepared from a dromedary immunized against DARC N-terminal extracellular domain and selected for DARC binding. A described VHH, CA52, does recognize native DARC on cells. It inhibits P. vivax invasion of erythrocytes and displaces interleukin-8 bound to DARC. The targeted epitope overlaps the well-defined DARC Fy6 epitope. K (D) of CA52-DARC equilibrium is sub-nanomolar, hence ideal to develop diagnostic or therapeutic compounds. Immunocapture by immobilized CA52 yielded highly purified DARC from engineered K562 cells. This first report on a VHH with specificity for a red blood cell protein exemplifies VHHs' potentialities to target, to purify, and to modulate the function of cellular markers.

Heterogeneous Phenotype of Long QT Syndrome Caused by the KCNH2-H562R Mutation: Importance of Familial Genetic Testing.

PubMed

Muñoz-Esparza, Carmen; García-Molina, Esperanza; Salar-Alcaraz, Mariela; Peñafiel-Verdú, Pablo; Sánchez-Muñoz, Juan J; Martínez Sánchez, Juan; Cabañas-Perianes, Valentín; Valdés Chávarri, Mariano; García Alberola, Arcadio; Gimeno-Blanes, Juan R

2015-10-01

Long QT syndrome is an inherited ion channelopathy that leads to syncope and sudden death. Because of the heterogeneous phenotype of this disease, genetic testing is fundamental to detect individuals with concealed long QT syndrome. In this study, we determined the features of a family with 13 carriers of the KCNH2-H562R missense mutation, which affects the pore region of the HERG channel. We identified the KCNH2-H562R mutation in a 65-year-old man with a prolonged QTc interval who had experienced an episode of torsade de pointes. Subsequently, a total of 13 mutation carriers were identified in the family. Carriers (age 48 [26] years; 46% males) underwent clinical evaluation, electrocardiography and echocardiography. The mean (standard deviation) QTc in carriers was 493 (42) ms (3 [23%] showed normal QTc); 6 (46%) had symptoms (4, syncope; 1, sudden death; 1, aborted sudden death [proband]). While under treatment with beta-blockers, 11 of 12 carriers (92%) remained asymptomatic at 5 years of follow-up (1 patient required left cardiac sympathectomy). The QTc shortening with beta-blockers was 50 (37) ms. There was 1 sudden death in a patient who refused treatment. Family study is essential in the interpretation of a genetic testing result. This article describes the heterogeneous and variable phenotype of a large family with the KCNH2-H562R mutation and highlights the role of genetic study for the appropriate identification of at-risk individuals who would benefit from treatment. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

Effect of 5-aminolevulinic acid on erythropoiesis: A preclinical in vitro characterization for the treatment of congenital sideroblastic anemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Tohru; Department of Molecular Hematology/Oncology, Tohoku University Graduate School, Sendai; Okamoto, Koji

2014-11-07

Highlights: • Treatment with ALA induces erythroid differentiation of K562 cells. • Transportation of ALA into erythroid cells occurs predominantly via SLC36A1. • ALA restores defects in ALAS2 in human iPS cell-derived erythroblasts. • ALA may represent a novel therapeutic option for CSA caused by ALAS2 mutations. - Abstract: Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), amore » crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was

Identification of pyrogallol as an antiproliferative compound present in extracts from the medicinal plant Emblica officinalis: effects on in vitro cell growth of human tumor cell lines.

PubMed

Khan, Mahmud Tareq Hassan; Lampronti, Ilaria; Martello, Dino; Bianchi, Nicoletta; Jabbar, Shaila; Choudhuri, Mohammad Shahabuddin Kabir; Datta, Bidduyt Kanti; Gambari, Roberto

2002-07-01

In this study we compared the in vitro antiproliferative activity of extracts from medicinal plants toward human tumor cell lines, including human erythromyeloid K562, B-lymphoid Raji, T-lymphoid Jurkat, erythroleukemic HEL cell lines. Extracts from Emblica officinalis were the most active in inhibiting in vitro cell proliferation, after comparison to those from Terminalia arjuna, Aphanamixis polystachya, Oroxylum indicum, Cuscuta reflexa, Aegle marmelos, Saraca asoka, Rumex maritimus, Lagerstroemia speciosa, Red Sandalwood. Emblica officinalis extracts have been studied previously, due to their hepatoprotective, antioxidant, antifungal, antimicrobial and anti-inflammatory medicinal activities. Gas chromatography/mass spectrometry analyses allowed to identify pyrogallol as the common compound present both in unfractionated and n-butanol fraction of Emblica officinalis extracts. Antiproliferative effects of pyrogallol were therefore determined on human tumor cell lines thus identifying pyrogallol as an active component of Emblica officinalis extracts.

Localization of the human 64kD autoantigen D1 to myofibrils in a subset of extraocular muscle fibers

NASA Technical Reports Server (NTRS)

Conley, C. A.; Fowler, V. M.

1999-01-01

PURPOSE. To evaluate the tissue-specific expression pattern of the 64kD human autoantigen D1, a tropomodulin-related protein that may be involved in thyroid-associated ophthalmopathy. METHODS. Recombinant 64kD human autoantigen D1 was generated in a bacterial expression system and used to immunize rabbits. Specific antibodies were affinity-purified and used for Western blots on normal and hyperthyroid rat and rabbit tissue, and immunofluorescence localization on cryosections of rat tissue. RESULTS. Anti-64kD human autoantigen D1 antibodies recognize specifically a approximately 70kD polypeptide in western blots of extraocular muscle, sternothyroid muscle, and smooth muscle. Immunofluorescence staining demonstrates that the 64kD human autoantigen D1 localizes to myofibrils in slow fibers from rat extraocular and sternothyroid muscle. The level of this protein is not altered in extraocular muscles from hyperthyroid rabbits. CONCLUSIONS. The 64kD human autoantigen D1 is expressed in slow fibers of extraocular and sternothyroid muscles as a component of myofibrils, and is not upregulated in conditions of hyperthyroidism.

Escherichia coli K1-induced cytopathogenicity of human brain microvascular endothelial cells.

PubMed

Khan, Naveed Ahmed; Iqbal, Junaid; Siddiqui, Ruqaiyyah

2012-01-01

Pathophysiology of Escherichia coli sepsis is complex involving circulating bacterial products, cytokine release, and sustained bacteremia resulting in the damage of vascular endothelium. Here, it is shown that E. coli K1 produced cytopathogenicity of human brain microvascular endothelial cells (HBMEC), that constitute the blood-brain barrier. Whole bacteria or their conditioned medium produced severe HBMEC damage suggesting E. coli K1-cytopathogenicity is a contact-independent process. Using lipopolysaccharide (LPS) inhibitor, polymyxin B, purified LPS extracted from E. coli K1 as well as LPS mutant derived from E. coli K1, we showed that LPS is not the sole determinant of E. coli K1-mediated HBMEC death. Bacterial product(s) for HBMEC cytopathogenicity was heat-labile suggesting LPS-associated proteins. Several isogenic gene-deletion mutants (ΔompA, ΔibeA, ΔibeB, Δcnf1) exhibited HBMEC cytopathogenicity similar to that produced by wild type E. coli K1. E. coli K1-mediated HBMEC death was independent of phosphatidylinositol 3-kinase (PI3K) but dependent partially on focal adhesion kinase (FAK) using HBMEC expressing dominant negative FAK and PI3K. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Na+/K+ pump is an important modulator of refractoriness and rotor dynamics in human atrial tissue.

PubMed

Sánchez, Carlos; Corrias, Alberto; Bueno-Orovio, Alfonso; Davies, Mark; Swinton, Jonathan; Jacobson, Ingemar; Laguna, Pablo; Pueyo, Esther; Rodríguez, Blanca

2012-03-01

Pharmacological treatment of atrial fibrillation (AF) exhibits limited efficacy. Further developments require a comprehensive characterization of ionic modulators of electrophysiology in human atria. Our aim is to systematically investigate the relative importance of ionic properties in modulating excitability, refractoriness, and rotor dynamics in human atria before and after AF-related electrical remodeling (AFER). Computer simulations of single cell and tissue atrial electrophysiology were conducted using two human atrial action potential (AP) models. Changes in AP, refractory period (RP), conduction velocity (CV), and rotor dynamics caused by alterations in key properties of all atrial ionic currents were characterized before and after AFER. Results show that the investigated human atrial electrophysiological properties are primarily modulated by maximal value of Na(+)/K(+) pump current (G(NaK)) as well as conductances of inward rectifier potassium current (G(K1)) and fast inward sodium current (G(Na)). G(NaK) plays a fundamental role through both electrogenic and homeostatic modulation of AP duration (APD), APD restitution, RP, and reentrant dominant frequency (DF). G(K1) controls DF through modulation of AP, APD restitution, RP, and CV. G(Na) is key in determining DF through alteration of CV and RP, particularly in AFER. Changes in ionic currents have qualitatively similar effects in control and AFER, but effects are smaller in AFER. The systematic analysis conducted in this study unravels the important role of the Na(+)/K(+) pump current in determining human atrial electrophysiology.

Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

NASA Astrophysics Data System (ADS)

Feuser, Paulo Emilio; Jacques, Amanda Virtuoso; Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin; dos Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H. Hermes

2016-04-01

The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

A 92-kDa human immunostimulatory protein.

PubMed Central

Fontan, E; Briend, E; Saklani-Jusforgues, H; d'Alayer, J; Vandekerckhove, J; Fauve, R M

1994-01-01

We purified to apparent homogeneity a human urinary glycoprotein of 92 kDa (HGP.92) that, administered intravenously at 250 micrograms/kg, fully protected mice against a lethal inoculum of Listeria monocytogenes. Since HGP.92 protected scid mice, which lack B and T lymphocytes, this increased resistance to Listeria did not appear to be lymphocyte mediated. Furthermore, inflammatory macrophages incubated with 6 nM HGP.92 inhibited the growth of Lewis carcinoma cells in vitro. These two activities appeared to depend on an oligosaccharide moiety, as they were lost after N-Glycanase treatment of HGP.92. Thus, the biological activity of HGP.92 was in some way related to a glycan moiety. Images PMID:8078887

Regulation of the Human Endogenous Retrovirus K (HML-2) Transcriptome by the HIV-1 Tat Protein

PubMed Central

Gonzalez-Hernandez, Marta J.; Cavalcoli, James D.; Sartor, Maureen A.; Contreras-Galindo, Rafael; Meng, Fan; Dai, Manhong; Dube, Derek; Saha, Anjan K.; Gitlin, Scott D.; Omenn, Gilbert S.; Kaplan, Mark H.

2014-01-01

ABSTRACT Approximately 8% of the human genome is made up of endogenous retroviral sequences. As the HIV-1 Tat protein activates the overall expression of the human endogenous retrovirus type K (HERV-K) (HML-2), we used next-generation sequencing to determine which of the 91 currently annotated HERV-K (HML-2) proviruses are regulated by Tat. Transcriptome sequencing of total RNA isolated from Tat- and vehicle-treated peripheral blood lymphocytes from a healthy donor showed that Tat significantly activates expression of 26 unique HERV-K (HML-2) proviruses, silences 12, and does not significantly alter the expression of the remaining proviruses. Quantitative reverse transcription-PCR validation of the sequencing data was performed on Tat-treated PBLs of seven donors using provirus-specific primers and corroborated the results with a substantial degree of quantitative similarity. IMPORTANCE The expression of HERV-K (HML-2) is tightly regulated but becomes markedly increased following infection with HIV-1, in part due to the HIV-1 Tat protein. The findings reported here demonstrate the complexity of the genome-wide regulation of HERV-K (HML-2) expression by Tat. This work also demonstrates that although HERV-K (HML-2) proviruses in the human genome are highly similar in terms of DNA sequence, modulation of the expression of specific proviruses in a given biological situation can be ascertained using next-generation sequencing and bioinformatics analysis. PMID:24872592

Lin28-let7 Modulates Radiosensitivity of Human Cancer Cells With Activation of K-Ras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Jee-Sun.; Kim, Jae-Jin; Byun, Ju-Yeon

2010-01-15

Purpose: To evaluate the potential of targeting Lin28-let7 microRNA regulatory network for overcoming the radioresistance of cancer cells having activated K-Ras signaling. Methods and Materials: A549 lung carcinoma cells and ASPC1 pancreatic cancer cells possessing K-RAS mutation were transfected with pre-let7a microRNA or Lin28 siRNA, respectively. Clonogenic assay, quantitative reverse transcription polymerase chain reaction, and Western analysis were performed. The effects of Lin28 on SQ20B cells having wild-type K-RAS, and a normal fibroblast were also assessed. Results: The overexpression of let-7a decreased expression of K-Ras and radiosensitized A549 cells. Inhibition of Lin28, a repressor of let-7, attenuated K-Ras expression andmore » radiosensitized A549 and ASPC1 cells. Neither SQ20B cells expressing wild-type K-RAS nor HDF, the normal human fibroblasts, were radiosensitized by this approach. Conclusions: The Lin28-let7 regulatory network may be a potentially useful therapeutic target for overcoming the radioresistance of human cancers having activated K-Ras signaling.« less

The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendelsohn, M.E.; Yan Zhu; O'Neill, S.

Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less

Corrective Action Plan for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

DOE Office of Scientific and Technical Information (OSTI.GOV)

NSTec Environmental Restoration

2011-04-30

This Corrective Action Plan has been prepared for Corrective Action Unit (CAU) 562, Waste Systems, in accordance with the Federal Facility Agreement and Consent Order (1996; as amended March 2010). CAU 562 consists of 13 Corrective Action Sites (CASs) located in Areas 2, 23, and 25 of the Nevada National Security Site. Site characterization activities were performed in 2009 and 2010, and the results are presented in Appendix A of the Corrective Action Decision Document for CAU 562. The scope of work required to implement the recommended closure alternatives is summarized. (1) CAS 02-26-11, Lead Shot, will be clean closedmore » by removing shot. (2) CAS 02-44-02, Paint Spills and French Drain, will be clean closed by removing paint and contaminated soil. As a best management practice (BMP), asbestos tile will be removed. (3) CAS 02-59-01, Septic System, will be clean closed by removing septic tank contents. As a BMP, the septic tank will be removed. (4) CAS 02-60-01, Concrete Drain, contains no contaminants of concern (COCs) above action levels. No further action is required; however, as a BMP, the concrete drain will be removed. (5) CAS 02-60-02, French Drain, was clean closed. Corrective actions were completed during corrective action investigation activities. As a BMP, the drain grates and drain pipe will be removed. (6) CAS 02-60-03, Steam Cleaning Drain, will be clean closed by removing contaminated soil. As a BMP, the steam cleaning sump grate and outfall pipe will be removed. (7) CAS 02-60-04, French Drain, was clean closed. Corrective actions were completed during corrective action investigation activities. (8) CAS 02-60-05, French Drain, will be clean closed by removing contaminated soil. (9) CAS 02-60-06, French Drain, contains no COCs above action levels. No further action is required. (10) CAS 02-60-07, French Drain, requires no further action. The french drain identified in historical documentation was not located during corrective action

Label-free image-based detection of drug resistance with optofluidic time-stretch microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kobayashi, Hirofumi; Lei, Cheng; Mao, Ailin; Jiang, Yiyue; Guo, Baoshan; Ozeki, Yasuyuki; Goda, Keisuke

2017-02-01

Acquired drug resistance is a fundamental predicament in cancer therapy. Early detection of drug-resistant cancer cells during or after treatment is expected to benefit patients from unnecessary drug administration and thus play a significant role in the development of a therapeutic strategy. However, the development of an effective method of detecting drug-resistant cancer cells is still in its infancy due to their complex mechanism in drug resistance. To address this problem, we propose and experimentally demonstrate label-free image-based drug resistance detection with optofluidic time-stretch microscopy using leukemia cells (K562 and K562/ADM). By adding adriamycin (ADM) to both K562 and K562/ADM (ADM-resistant K562 cells) cells, both types of cells express unique morphological changes, which are subsequently captured by an optofluidic time-stretch microscope. These unique morphological changes are extracted as image features and are subjected to supervised machine learning for cell classification. We hereby have successfully differentiated K562 and K562/ADM solely with label-free images, which suggests that our technique is capable of detecting drug-resistant cancer cells. Our optofluidic time-stretch microscope consists of a time-stretch microscope with a high spatial resolution of 780 nm at a 1D frame rate of 75 MHz and a microfluidic device that focuses and orders cells. We compare various machine learning algorithms as well as various concentrations of ADM for cell classification. Owing to its unprecedented versatility of using label-free image and its independency from specific molecules, our technique holds great promise for detecting drug resistance of cancer cells for which its underlying mechanism is still unknown or chemical probes are still unavailable.

Antigenic and functional properties of the human red blood cell urea transporter hUT-B1.

PubMed

Lucien, Nicole; Sidoux-Walter, Frédéric; Roudier, Nathalie; Ripoche, Pierre; Huet, Martine; Trinh-Trang-Tan, Marie-Marcelle; Cartron, Jean-Pierre; Bailly, Pascal

2002-09-13

The Kidd (JK) blood group locus encodes the urea transporter hUT-B1, which is expressed on human red blood cells and other tissues. The common JK*A/JK*B blood group polymorphism is caused by a single nucleotide transition G838A changing Asp-280 to Asn-280 on the polypeptide, and transfection of erythroleukemic K562 cells with hUT-B1 cDNAs carrying either the G838 or the A838 nucleotide substitutions resulted in the isolation of stable clones that expressed the Jk(a) or Jk(b) antigens, respectively, thus providing the first direct demonstration that the hUT-B1 gene encodes the Kidd blood group antigens. In addition, immunochemical analysis of red blood cells demonstrated that hUT-B1 also exhibits ABO determinants attached to the single N-linked sugar chain at Asn-211. Moreover, immunoadsorption studies, using inside-out and right-side-out red cell membrane vesicles as competing antigen, demonstrated that the C- and N-terminal ends of hUT-B1 are oriented intracellularly. Mutagenesis and functional studies by expression in Xenopus oocytes revealed that both cysteines Cys-25 and Cys-30 (but not alone) are essential for plasma membrane addressing. Conversely, the transport function was not affected by the JK*A/JK*B polymorphism, C-terminal deletion (residues 360-389), or mutation of the extracellular N-glycosylation consensus site and remains poorly para-chloromercuribenzene sulfonate (pCMBS)-sensitive. However, transport studies by stopped flow light scattering using Jk-K562 transfectants demonstrated that the hUT-B1-mediated urea transport is pCMBS-sensitive in an erythroid context, as reported previously for the transporter of human red blood cells. Mutagenesis analysis also indicated that Cys-151 and Cys-236, at least alone, are not involved in pCMBS inhibition. Altogether, these antigenic, topologic, and functional properties might have implications into the physiology of hUT-B1 and other members of the urea transporter family.

RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Houcai; Yu, Jing; Zhang, Lixia

2014-04-18

Highlights: • RPS27a expression was up-regulated in advanced-phase CML and AL patients. • RPS27a knockdown changed biological property of K562 and K562/G01 cells. • RPS27a knockdown affected Raf/MEK/ERK, P21 and BCL-2 signaling pathways. • RPS27a knockdown may be applicable for new combination therapy in CML patients. - Abstract: Ribosomal protein S27a (RPS27a) could perform extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. The high expression level of RPS27a was reported in solid tumors, and we found that the expression level of RPS27a was up-regulated in advanced-phase chronic myeloid leukemia (CML) and acute leukemia (AL)more » patients. In this study, we explored the function of RPS27a in leukemia cells by using CML cell line K562 cells and its imatinib resistant cell line K562/G01 cells. It was observed that the expression level of RPS27a was high in K562 cells and even higher in K562/G01 cells. Further analysis revealed that RPS27a knockdown by shRNA in both K562 and K562G01 cells inhibited the cell viability, induced cell cycle arrest at S and G2/M phases and increased cell apoptosis induced by imatinib. Combination of shRNA with imatinib treatment could lead to more cleaved PARP and cleaved caspase-3 expression in RPS27a knockdown cells. Further, it was found that phospho-ERK(p-ERK) and BCL-2 were down-regulated and P21 up-regulated in RPS27a knockdown cells. In conclusion, RPS27a promotes proliferation, regulates cell cycle progression and inhibits apoptosis of leukemia cells. It appears that drugs targeting RPS27a combining with tyrosine kinase inhibitor (TKI) might represent a novel therapy strategy in TKI resistant CML patients.« less

Vaccination directed against the human endogenous retrovirus-K (HERV-K) gag protein slows HERV-K gag expressing cell growth in a murine model system.

PubMed

Kraus, Benjamin; Fischer, Katrin; Sliva, Katja; Schnierle, Barbara S

2014-03-26

Human endogenous retroviruses (HERVs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. However enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed inter-alia in HIV-infected individuals and tumor patients. Therefore HERV-K might serve as a tumor-specific antigen or even as a constant target for the development of an HIV vaccine. To verify our hypothesis, we tested the immunogenicity of HERV-K Gag by using a recombinant vaccinia virus (MVA-HKcon) expressing the HERV-K Gag protein and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) and the HERV-K Gag protein (RLZ-HKGag cells). Subcutaneous application of RLZ-HKGag cells into syngenic BALB/c mice resulted in the formation of local tumors in MVA vaccinated mice. MVA-HKcon vaccination reduced the tumor growth. Furthermore, intravenous injection of RLZ-HKGag cells led to the formation of pulmonary metastases. Vaccination of tumor-bearing mice with MVA-HKcon drastically reduced the number of pulmonary RLZ-HKGag tumor nodules compared to vaccination with wild-type MVA. The data demonstrate that HERV-K Gag is a useful target for vaccine development and might offer new treatment opportunities for cancer patients.

Analysis pipelines and packages for Infinium HumanMethylation450 BeadChip (450k) data

PubMed Central

Morris, Tiffany J.; Beck, Stephan

2015-01-01

The Illumina HumanMethylation450 BeadChip has become a popular platform for interrogating DNA methylation in epigenome-wide association studies (EWAS) and related projects as well as resource efforts such as the International Cancer Genome Consortium (ICGC) and the International Human Epigenome Consortium (IHEC). This has resulted in an exponential increase of 450k data in recent years and triggered the development of numerous integrated analysis pipelines and stand-alone packages. This review will introduce and discuss the currently most popular pipelines and packages and is particularly aimed at new 450k users. PMID:25233806

Basolateral K+ channel involvement in forskolin-activated chloride secretion in human colon

PubMed Central

McNamara, Brian; Winter, Desmond C; Cuffe, John E; O'Sullivan, Gerald C; Harvey, Brian J

1999-01-01

In this study we investigated the role of basolateral potassium transport in maintaining cAMP-activated chloride secretion in human colonic epithelium. Ion transport was quantified in isolated human colonic epithelium using the short-circuit current technique. Basolateral potassium transport was studied using nystatin permeabilization. Intracellular calcium measurements were obtained from isolated human colonic crypts using fura-2 spectrofluorescence imaging. In intact isolated colonic strips, forskolin and prostaglandin E2 (PGE2) activated an inward transmembrane current (ISC) consistent with anion secretion (for forskolin ΔISC = 63.8 ± 6.2 μA cm−2, n = 6; for PGE2 ΔISC = 34.3 ± 5.2 μA cm−2, n = 6). This current was inhibited in chloride-free Krebs solution or by inhibiting basolateral chloride uptake with bumetanide and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The forskolin- and PGE2-induced chloride secretion was inhibited by basolateral exposure to barium (5 mM), tetrapentylammonium (10 μM) and tetraethylammonium (10 mM). The transepithelial current produced under an apical to serosal K+ gradient in nystatin-perforated colon is generated at the basolateral membrane by K+ transport. Forskolin failed to activate this current under conditions of high or low calcium and failed to increase the levels of intracellular calcium in isolated crypts In conclusion, we propose that potassium recycling through basolateral K+ channels is essential for cAMP-activated chloride secretion. PMID:10432355

All human Na(+)-K(+)-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides.

PubMed

Wang, J; Velotta, J B; McDonough, A A; Farley, R A

2001-10-01

Three alpha-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (K(d)) for all samples calculated using the single-site model was 18 +/- 6 nM (mean +/- SD). No difference in K(d) was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only approximately 50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha-isoforms are similar to each other, although calculated K(d) values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

Cytotoxicity and Antiproliferative Activity Assay of Clove Mistletoe (Dendrophthoe pentandra (L.) Miq.) Leaves Extracts

PubMed Central

Elsyana, Vida; Bintang, Maria; Priosoeryanto, Bambang Pontjo

2016-01-01

Clove mistletoe (Dendrophthoe pentandra (L.) Miq.) is a semiparasitic plant that belongs to Loranthaceae family. Clove mistletoe was traditionally used for cancer treatment in Indonesia. In the present study, we examined cytotoxicity of clove mistletoe leaves extracts against brine shrimps and conducted their antiproliferative activity on K562 (human chronic myelogenous leukemia) and MCM-B2 (canine benign mixed mammary) cancer cell lines in vitro. The tested samples were water extract, ethanol extract, ethanol fraction, ethyl acetate fraction, and n-hexane fraction. Cytotoxicity was screened using Brine Shrimp Lethality Test (BSLT). Antiproliferative activity was conducted using Trypan Blue Dye Method and cells were counted using haemocytometer. The results showed that n-hexane fraction exhibited significant cytotoxicity with LC50 value of 55.31 μg/mL. The n-hexane fraction was then considered for further examination. The n-hexane fraction of clove mistletoe could inhibit growth of K562 and MCM-B2 cancer cell lines in vitro. The inhibition activity of clove mistletoe n-hexane fraction at concentration of 125 μg/mL on K562 cancer cell lines was 38.69%, while on MCM-B2 it was 41.5%. Therefore, it was suggested that clove mistletoe had potential natural anticancer activity. PMID:27099614

Antiradical and cytotoxic activity of different Helichrysum plicatum flower extracts.

PubMed

Bigović, Dubravka; Savikin, Katarina; Janković, Teodora; Menković, Nebojsa; Zdunić, Gordana; Stanojković, Tatjana; Djurić, Zorica

2011-06-01

Flowers of Helichrysum plicatum were extracted under different experimental conditions, and their antioxidant activity was determined by DPPH radical scavenging assay. Extracts obtained with higher concentration of ethyl acetate (90% or 100%) were found to contain the greatest amount of total phenolics (> 250 mg gallic acid equivalents/g of dried extract), and high correlation between total phenolic content and antiradical activity was observed (r = -0.79). Based on the total phenolic content and antiradical activity, some extracts were selected for investigation of cytotoxic activity toward PC3, HeLa and K562 human cancer cell lines in vitro. All tested extracts exhibited moderate activity against HeLa cells (41.9-42.1 microg/mL), whereas the extract obtained with 100% ethyl acetate was the most active against K562 and PC3 cell lines (25.9 and 39.2 microg/mL, respectively). Statistical analysis revealed significant correlation between total phenolic content and cytotoxic activity against PC3 and K562 cells. HPLC identification of phenolic compounds from the extracts indicated the presence of apigenin, naringenin and kaempferol as free aglycones, and glycosides of apigenin, naringenin, quercetin and kaempferol. Among aglycones, kaempferol displayed moderate cytostatic activity against all cell lines (24.8-64.7 microM).

Interaction of External K, Na, and Cardioactive Steroids with the Na-K Pump of the Human Red Blood Cell

PubMed Central

Sachs, John R.

1974-01-01

The interaction of extracellular Na (Nao), K (Ko), and strophanthidin with the Na-K pump of the human red blood cell has been investigated. Inhibition by submaximal concentrations of strophanthidin rapidly reaches a level which does not increase further over a relatively long period of time. Under these circumstances, it is possible to apply a steady-state kinetic analysis to the interaction of Nao, Ko, and strophanthidin with the pump. In Na-free solutions, strophanthidin increases the apparent K1/2 of the pump for Ko, but does not change the form of the relation between the reciprocal of the active K influx (i M K P–1) and the reciprocal of [Ko] ([Ko]–1); the relation both in the presence and absence of strophanthidin is adequately described by a straight line. In solutions containing Na, strophanthidin changes the form of the curve describing the relation between i M K P–1 vs. [Ko]–1; the curve becomes more parabolic in solutions containing strophanthidin. The rate of ouabain binding to K-free cells has also been measured; in the absence of K, the rate of binding is unaffected by Nao. The data are considered in terms of a simple kinetic model. The findings can be explained if it is supposed that at low external K the form of the pump combined with one Nao is more likely to combine with strophanthidin than is the uncombined form of the pump. The uncombined form of the pump is more likely to combine with K even at very low Ko than with strophanthidin. PMID:4812632

Effect of orthodontic force on the expression of PI3K, Akt, and P70S6 K in the human periodontal ligament during orthodontic loading.

PubMed

Xu, Yunhe; Shen, Jiayuan; Muhammed, Fenik Kaml; Zheng, Bowen; Zhang, Yuejiao; Liu, Yi

2017-10-01

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine protein kinases involved in the regulation of cell growth, proliferation, and differentiation through the PI3K/Akt/mTOR/P70S6 K signalling pathway. P70S6 K as a downstream molecule of mTOR is activated by phosphorylation and subsequently promotes the synthesis of ribosomal and translational proteins. In this study, we investigated the role of PI3K, Akt, and P70S6 K in human periodontal tissue remodelling during orthodontic loading. The prepared tissue specimens taken from 4 extracted premolars were processed for immunolabelling. The changes in the expression of PI3K, Akt, and P70S6 K in the periodontal tissues were detected by real-time quantitative-polymerase chain reaction and Western blot analysis. The results from real-time quantitative-polymerase chain reaction and Western blot both showed that the expression of PI3K, Akt, and P70S6 K in the experimental group began to increase at 3 days and increased significantly at 10 days, then decreased approaching the control group level at 28 days. Our findings showed that the expression of PI3K, Akt, and P70S6 K in human periodontal ligament demonstrated a variability during the orthodontic loading, which suggested that the PI3K/Akt/mTOR/P70S6 K signal pathway was involved in orthodontic tooth movement and played a role in the process of periodontium remodelling. Copyright © 2017 John Wiley & Sons, Ltd.

Analysis pipelines and packages for Infinium HumanMethylation450 BeadChip (450k) data.

PubMed

Morris, Tiffany J; Beck, Stephan

2015-01-15

The Illumina HumanMethylation450 BeadChip has become a popular platform for interrogating DNA methylation in epigenome-wide association studies (EWAS) and related projects as well as resource efforts such as the International Cancer Genome Consortium (ICGC) and the International Human Epigenome Consortium (IHEC). This has resulted in an exponential increase of 450k data in recent years and triggered the development of numerous integrated analysis pipelines and stand-alone packages. This review will introduce and discuss the currently most popular pipelines and packages and is particularly aimed at new 450k users. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Escherichia coli K1 invasion of human brain microvascular endothelial cells.

PubMed

Loh, Lip Nam; Ward, Theresa H

2012-01-01

The pathogenic Escherichia coli strain E. coli K1 is a primary causative agent of neonatal meningitis. Understanding how these bacteria cross the blood-brain barrier is vital to develop therapeutics. Here, we describe the use of live-cell imaging techniques to study E. coli K1 interactions with cellular markers following infection of human brain microvascular endothelial cells, a model system of the blood-brain barrier. We also discuss optimization of endothelial cell transfection conditions using nonviral transfection technique, bacterial labeling techniques, and in vitro assays to screen for fluorescent bacteria that retain their ability to invade host cells. Copyright © 2012 Elsevier Inc. All rights reserved.

JS-K promotes apoptosis by inducing ROS production in human prostate cancer cells.

PubMed

Qiu, Mingning; Chen, Lieqian; Tan, Guobin; Ke, Longzhi; Zhang, Sai; Chen, Hege; Liu, Jianjun

2017-03-01

Reactive oxygen species (ROS) are chemical species that alter redox status, and are responsible for inducing carcinogenesis. The purpose of the present study was to assess the effects of the glutathione S transferase-activated nitric oxide donor prodrug, JS-K, on ROS accumulation and on proliferation and apoptosis in human prostate cancer cells. Cell proliferation and apoptosis, ROS accumulation and the activation of the mitochondrial signaling pathway were measured. The results demonstrated that JS-K may inhibit prostate cancer cell growth in a dose- and time-dependent manner, and induce ROS accumulation and apoptosis in a dose-dependent manner. With increasing concentrations of JS-K, expression of pro-apoptotic proteins increased, but Bcl-2 expression decreased. Additionally, the antioxidant N-acetylcysteine reversed JS-K-induced cell apoptosis; conversely, the pro-oxidant glutathione disulfide exacerbated JS-K-induced apoptosis. In conclusion, the data suggest that JS-K induces prostate cancer cell apoptosis by increasing ROS levels.

JS-K promotes apoptosis by inducing ROS production in human prostate cancer cells

PubMed Central

Qiu, Mingning; Chen, Lieqian; Tan, Guobin; Ke, Longzhi; Zhang, Sai; Chen, Hege; Liu, Jianjun

2017-01-01

Reactive oxygen species (ROS) are chemical species that alter redox status, and are responsible for inducing carcinogenesis. The purpose of the present study was to assess the effects of the glutathione S transferase-activated nitric oxide donor prodrug, JS-K, on ROS accumulation and on proliferation and apoptosis in human prostate cancer cells. Cell proliferation and apoptosis, ROS accumulation and the activation of the mitochondrial signaling pathway were measured. The results demonstrated that JS-K may inhibit prostate cancer cell growth in a dose- and time-dependent manner, and induce ROS accumulation and apoptosis in a dose-dependent manner. With increasing concentrations of JS-K, expression of pro-apoptotic proteins increased, but Bcl-2 expression decreased. Additionally, the antioxidant N-acetylcysteine reversed JS-K-induced cell apoptosis; conversely, the pro-oxidant glutathione disulfide exacerbated JS-K-induced apoptosis. In conclusion, the data suggest that JS-K induces prostate cancer cell apoptosis by increasing ROS levels. PMID:28454225

Vitamins C and K3 sensitize human urothelial tumors to gemcitabine.

PubMed

Kassouf, Wassim; Highshaw, Ralph; Nelkin, Gina M; Dinney, Colin P; Kamat, Ashish M

2006-10-01

We evaluated the antitumor effects of vitamins C and K3 for human urothelial carcinoma and the potential use of the combination of vitamins C plus K3 as a sensitizing agent for conventional chemotherapy for urothelial carcinoma. The antiproliferative and apoptotic effects of vitamin C alone, vitamin K3 alone, vitamins C plus K3, gemcitabine alone and gemcitabine plus vitamins C plus K3 were assessed in vitro by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, propidium iodide staining and flow cytometry. For in vivo studies we implanted UMUC-14 tumorigenic urothelial carcinoma cells into the subcutis of nude mice. One week later we treated 10 mice each with saline (control), vitamins C plus K3, gemcitabine or gemcitabine plus vitamins C plus K3. Treatment was continued for 4 weeks, followed by necropsy. Tumor volume was measured and tumor kinetics were established. Apoptosis and proliferation were evaluated in tumor sections using immunohistochemistry and TUNEL assay. Vitamins C plus K3 induced cytostasis and caused apoptosis to a greater degree than either vitamin alone (p < 0.05). Vitamins C plus K3 also substantially augmented the effects of gemcitabine in vitro. There were 32.3% apoptosis with gemcitabine plus vitamins C plus K3, 5.3% with gemcitabine alone and 15.8% with vitamins C plus K3 alone (p < 0.05). In vivo tumor growth was substantially inhibited by gemcitabine plus vitamins C plus K3 compared with that in the control or for either agent alone. Mean tumor weight and growth rate in the gemcitabine plus vitamins C plus K3 group (237 mg and 11.3 mm3 daily) were decreased compared with those in the control (530 mg and 34.3 mm3 daily), and those for vitamins C plus K3 alone (490 mg and 25.2 mm3 daily) and gemcitabine alone (400 mg and 21.3 mm3 daily) (p < 0.05). Vitamins C and K3 have significant antiproliferative and apoptotic effects when used in combination. This combination enhances the efficacy of gemcitabine against bladder

Inhibitory effects of physalin B and physalin F on various human leukemia cells in vitro.

PubMed

Chiang, H C; Jaw, S M; Chen, P M

1992-01-01

Physalins B and F were isolated and characterized from the ethanolic extract of the whole plant of Physalis angulata L. (Solanaceae). Both physalin B and physalin F inhibited the growth of several human leukemia cells: K562 (erythroleukemia), APM1840 (acute T lymphoid leukemia), HL-60 (acute promyelocytic leukemia), KG-1 (acute myeloid leukemia), CTV1 (acute monocytic leukemia) and B cell (acute B lymphoid leukemia). Physalin F showed a stronger activity against these leukemia cells than physalin B, especially against acute myeloid leukemia (KG-1) and acute B lymphoid leukemia (B cell). From the structural features, the active site seems to be the functional epoxy group for physalin F and the double bond for physalin B located at carbon 5 and 6; the former is much more active than the latter as regards anti-leukemic effects.

Nitric oxide-releasing nanoparticles: synthesis, characterization, and cytotoxicity to tumorigenic cells

NASA Astrophysics Data System (ADS)

Pelegrino, Milena T.; Silva, Letícia C.; Watashi, Carolina M.; Haddad, Paula S.; Rodrigues, Tiago; Seabra, Amedea B.

2017-02-01

Nitric oxide (NO) is involved in several biological processes, including toxicity against tumor cells. The aim of this study was to synthesize, characterize, and evaluate the cytotoxicity of NO-releasing chitosan nanoparticles. A thiol-containing molecule, mercaptosuccinic acid (MSA), was encapsulated (encapsulation efficiency of 99%) in chitosan/sodium tripolyphosphate nanoparticles (CS NPs). The obtained nanoparticles showed an average hydrodynamic size of 108.40 ± 0.96 nm and polydispersity index of 0.26 ± 0.01. MSA-CS NPs were nitrosated leading to S-nitroso-MSA-CS NPs, which act as NO donor. The cytotoxicity of CS NPs, MSA-CS NPs, and S-nitroso-MSA-CS NPs were evaluated in several tumor cells, including human hepatocellular carcinoma (HepG2), mouse melanoma (B16F10), and human chronic myeloid leukemia (K562) cell lines and Lucena-1, a vincristine-resistant K562 cell line. Both CS NPs and MSA-CS NPs did not cause toxic effects in these cells, whereas S-nitroso-MSA-CS NPs caused potent cytotoxic effects in all the tested tumor cell lines. The half-maximal inhibitory concentration values of S-nitroso-MSA-CS NPs were 19.7, 10.5, 22.8, and 27.8 μg·mL-1 for HepG2, B16F10, K562, and Lucena-1 cells, respectively. In contrast, S-nitroso-MSA-CS NPs exhibited lower cytotoxic to non-tumorigenic melanocytes (Melan-A) when compared with melanoma B16F10. Therefore, the results highlight the potential use of NO-releasing CS NPs in antitumor chemotherapy.

Modeling and Docking Studies on Novel Mutants (K71L and T204V) of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

PubMed Central

Elengoe, Asita; Naser, Mohammed Abu; Hamdan, Salehhuddin

2014-01-01

The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO) were mutated. 3D mutant models (K71L and T204V) using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255), as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy. PMID:24758925

Pertussis toxin inhibits somatostatin-induced K/sup +/ conductance in human pituitary tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamashita, N.; Kojima, I.; Shibuya, N.

1987-07-01

The effect of pertussis toxin on somatostatin-induced K/sup +/ current was examined in dissociated human pituitary tumor cells obtained from two acromegalic patients. Somatostatin-induced hyperpolarization or K/sup +/ current was observed in 20 of 23 cells in adenoma 1 and 10 of 11 cells in adenoma 2. After treatment with pertussis toxin for 24 h, these responses were completely suppressed (0/14 in adenoma, 1, 0/10 in adenoma 2). Spontaneous action potentials, K/sup +/, Na/sup +/, and Ca/sup 2 +/ currents were well preserved after pertussis toxin treatment. When crude membrane fraction was incubated with (/sup 32/P)NAD, a 41K protein wasmore » ADP-ribosylated by pertussis toxin. Hormone release was inhibited by somatostatin and this inhibition was blocked by pertussis toxin treatment.« less

Hematopoietic Stem Cell and Its Growth Factor

DTIC Science & Technology

1988-02-16

Bamberger and AS Felin . 1981. A multipotential leukemia cell line (K562) of human origin. Proc Soc Exp Biol Med 166:546. 40. Marie JP, CA Izaquirre, CI...at day 12 due to the degeneration of cells in the colonies. Monoclonal antibodies against human nonlymphoid leukemia cell lines which have...granulocyte mAb with acute myclocytic and myelomonocytic and lymphocytic leukemia ................................... 18 A-4 Antigen ML143 is expressed on

Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells.

PubMed

Lauf, Peter K; Misri, Sandeep; Chimote, Ameet A; Adragna, Norma C

2008-03-01

This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain +/- bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media approximately 90% of the total Rb influx occurred through the Na-K pump and NKCC and approximately 10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased K(c) by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 approximately 25 microM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.

Mutational definition of functional domains within the Rev homolog encoded by human endogenous retrovirus K.

PubMed

Bogerd, H P; Wiegand, H L; Yang, J; Cullen, B R

2000-10-01

Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the approximately 25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

Chemistry and Selective Tumor Cell Growth Inhibitory Activity of Polyketides from the South China Sea Sponge Plakortis sp.

PubMed

Li, Jiao; Li, Cui; Riccio, Raffaele; Lauro, Gianluigi; Bifulco, Giuseppe; Li, Tie-Jun; Tang, Hua; Zhuang, Chun-Lin; Ma, Hao; Sun, Peng; Zhang, Wen

2017-05-03

Simplextone E ( 1 ), a new metabolite of polyketide origin, was isolated with eight known analogues ( 2 - 9 ) from the South China Sea sponge Plakortis sp. The relative configuration of the new compound was elucidated by a detailed analysis of the spectroscopic data and quantum mechanical calculation of NMR chemical shifts, aided by the newly reported DP4+ approach. Its absolute configuration was determined by the TDDFT/ECD calculation. Simplextone E ( 1 ) is proven to be one of the isomers of simplextone D. The absolute configuration at C-8 in alkyl chain of plakortone Q ( 2 ) was also assigned based on the NMR calculation. In the preliminary in vitro bioassay, compounds 6 and 7 showed a selective growth inhibitory activity against HCT-116 human colon cancer cells with IC 50 values of 8.3 ± 2.4 and 8.4 ± 2.3 μM, corresponding to that of the positive control, adriamycin (IC 50 4.1 μM). The two compounds also showed selective activities towards MCF-7 human breast cancer and K562 human erythroleukemia cells while compound 3 only displayed weak activity against K562 cells.

Effect of phenazine methosulphate on K+ transport in human red cells.

PubMed

Gibson, John S; Muzyamba, Morris C; Ellory, Clive J

2003-01-01

The effect of phenazine methosulphate (PMS; 1 mM) on (86Rb+) K+ transport in human red cells was investigated to ascertain its action on the K+-Cl- cotransporter (KCC; defined as the Cl- dependent component of K+ flux measured in the presence of ouabain and bumetanide) and the Ca2+-activated K+ channel (Gardos channel; defined as the clotrimazole, 5 microM, -sensitive K+ flux). In the presence of Ca2+, both transport pathways were stimulated but effects were markedly greater under deoxygenated conditions (5-fold for KCC; 20-fold for the Gardos channel). KCC activation was inhibited by prior treatment with calyculin A (100 nM), implying action via protein dephosphorylation. Activation of the Gardos channel correlated with 28 +/- 3% inhibition of the plasma membrane Ca2+ pump, with maximal activity reduced from 7.7 +/- 1.1 to 2.7 +/- 0.7 micromol.(l cells.h)(-1) (all means +/- S.E.M. for n = 3), and a 3-fold increase in sensitivity of the channel to Ca2+ (EC50 reduced from 437 +/- 156 to 152 +/- 57 nM). Increased availability of NADH in deoxygenated conditions, resulting in increased free radical generation by PMS, may be responsible. We speculate that the similarity of the K+ transport phenotype produced by PMS to that seen in deoxygenated sickle cells is relevant to the pathophysiology of sickle cell disease. Copyright 2003 S. Karger AG, Basel

Design, Synthesis, and Biological Evaluation of Novel 1,3,4-Thiadiazole Derivatives as Potential Antitumor Agents against Chronic Myelogenous Leukemia: Striking Effect of Nitrothiazole Moiety

DOE PAGES

Altıntop, Mehlika; Ciftci, Halil; Radwan, Mohamed; ...

2017-12-27

In an attempt to develop potent antitumor agents, new 1,3,4-thiadiazole derivatives were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines, including the K562 chronic myelogenous leukemia cell line that expresses the Bcr-Abl tyrosine kinase. N-(5-Nitrothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide (2) inhibited the Abl protein kinase with an IC 50 value of 7.4 µM and showed selective activity against the Bcr-Abl positive K562 cell line. Furthermore, a Bcr-Abl-compound 2 molecular modelling simulation highlighted the anchoring role of the nitrothiazole moiety in bonding and hydrophobic interaction with the key amino acid residues. These results provide promising starting points for further developmentmore » of novel kinase inhibitors.« less

Design, Synthesis, and Biological Evaluation of Novel 1,3,4-Thiadiazole Derivatives as Potential Antitumor Agents against Chronic Myelogenous Leukemia: Striking Effect of Nitrothiazole Moiety

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altıntop, Mehlika; Ciftci, Halil; Radwan, Mohamed

In an attempt to develop potent antitumor agents, new 1,3,4-thiadiazole derivatives were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines, including the K562 chronic myelogenous leukemia cell line that expresses the Bcr-Abl tyrosine kinase. N-(5-Nitrothiazol-2-yl)-2-((5-((4-(trifluoromethyl)phenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetamide (2) inhibited the Abl protein kinase with an IC 50 value of 7.4 µM and showed selective activity against the Bcr-Abl positive K562 cell line. Furthermore, a Bcr-Abl-compound 2 molecular modelling simulation highlighted the anchoring role of the nitrothiazole moiety in bonding and hydrophobic interaction with the key amino acid residues. These results provide promising starting points for further developmentmore » of novel kinase inhibitors.« less

Evaluation and structure-activity relationship analysis of a new series of arylnaphthalene lignans as potential anti-tumor agents.

PubMed

Luo, Jiaoyang; Hu, Yichen; Kong, Weijun; Yang, Meihua

2014-01-01

Arylnaphthalene lignan lactones have attracted considerable interest because of their anti-tumor and anti-hyperlipidimic activities. However, to our knowledge, few studies have explored the effects of these compounds on human leukemia cell lines. In this study, five arylnaphthalene lignans including 6'-hydroxy justicidin A (HJA), 6'-hydroxy justicidin B (HJB), justicidin B (JB), chinensinaphthol methyl ether (CME) and Taiwanin E methyl ether (TEME) were isolated from Justicia procumbens and their effects on the proliferation and apoptosis of the human leukemia K562 cell line were investigated then used to assess structure-activity relationships. To achieve these aims, cytotoxicity was assayed using the MTT assay, while intracellular SOD activity was detected using the SOD Activity Assay kit. Apoptosis was measured by both the using a cycle TEST PLUS DNA reagent kit as well as the FITC Annexin V apoptosis detection kit in combination with flow cytometry. Activation of caspase-mediated apoptosis was evaluated using a FITC active Caspase-3 apoptosis kit and flow cytometry. The results indicated that HJB, HJA and JB significantly inhibited the growth of K562 cells by decreasing both proliferation and SOD activity and inducing apoptosis. The sequence of anti-proliferative activity induced by the five tested arylnaphthalenes by decreasing strength was HJB > HJA > JB > CME > TEME. HJB, HJA and JB also decreased SOD activity and induced apoptosis in a dose-dependent manner. Activation of caspase-3 further indicated that HJB, HJA and JB induced caspase-dependent intrinsic and/or extrinsic apoptosis pathways. Together, these assays suggest that arylnaphthalene lignans derived from Justicia procumbens induce apoptosis to varying degrees, through a caspase-dependent pathway in human leukemia K562 cells. Furthermore, analysis of structure-activity relationships suggest that hydroxyl substitution at C-1 and C-6' significantly increased the antiproliferative activity of

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

PubMed Central

Stiefelhagen, Marius; Sellner, Leopold; Kleinschmidt, Jürgen A; Jauch, Anna; Laufs, Stephanie; Wenz, Frederik; Zeller, W Jens; Fruehauf, Stefan; Veldwijk, Marlon R

2008-01-01

Background For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. Methods To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. Results Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. Conclusion Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors. PMID:18789140

The Recognition of N-Glycans by the Lectin ArtinM Mediates Cell Death of a Human Myeloid Leukemia Cell Line

PubMed Central

Carvalho, Fernanda Caroline; Soares, Sandro Gomes; Tamarozzi, Mirela Barros; Rego, Eduardo Magalhães; Roque-Barreira, Maria-Cristina

2011-01-01

ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment. PMID:22132163

THE INACTIVATION OF PENICILLINS F, G, K, AND X BY HUMAN AND RABBIT SERUM

PubMed Central

Eagle, Harry

1947-01-01

1. Penicillins F, G, K, and X were all inactivated by human and rabbit serum, but two qualitatively distinct mechanisms were apparently involved. 2. One was a slow inactivation of all four penicillins by a relatively thermostable serum component which was not demonstrably affected by heating for 60 minutes at 56°C. (a) In both human and rabbit serum this general inactivation of penicillin behaved like a pseudo first order reaction, with a velocity constant of 0.05–0.07 for penicillin X, and 0.09–0.11 for penicillins F and G. (b) The percentage of penicillins F, G, and X inactivated per hour was independent of their concentration over the range 0.4 to 50 micrograms per cc., averaging 9.5, 10, and 6.5 per cent, respectively, in human serum, and 9,8.5, and 5 per cent in rabbit serum. (c) The rate of inactivation varied linearly with the concentration of the serum factor. (d) Penicillin X was consistently and significantly less susceptible to inactivation than any of the other penicillins. Although minor differences were observed between F and G, these were not consistent, and are of questionable significance. 3. Superimposed on this slow inactivation of penicillins F, G, K, and X by a thermostable serum component was a much faster inactivation observed only with penicillin K. (a) In both rabbit and human serum, the serum factor responsible for this inactivation was highly thermolabile, and was almost completely destroyed within 5 minutes at 56°C., leaving only a thermostable component, not affected by further heating. (b) The inactivation of K by this thermolabile component was not a first order reaction, but varied with the concentration of both serum and penicillin. At high concentrations of K, the rate of inactivation due to the thermolabile factor was negligible, and penicillin K was destroyed no more rapidly than F, G, or X. The rate of inactivation increased as the concentration of penicillin was reduced. At penicillin K concentrations of 50, 10, 2, and 0

Localization and regulation of mouse pantothenate kinase 2 [The PanK2 Genes of Mouse and Human Specify Proteins with Distinct Subcellular Locations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leonardi, Roberta; Zhang, Yong-Mei; Lykidis, Athanasios

2007-09-07

Coenzyme A (CoA) biosynthesis is initiated by pantothenatekinase (PanK) and CoA levels are controlled through differentialexpression and feedback regulation of PanK isoforms. PanK2 is amitochondrial protein in humans, but comparative genomics revealed thatacquisition of a mitochondrial targeting signal was limited to primates.Human and mouse PanK2 possessed similar biochemical properties, withinhibition by acetylCoA and activation by palmitoylcarnitine. Mouse PanK2localized in the cytosol, and the expression of PanK2 was higher in humanbrain compared to mouse brain. Differences in expression and subcellularlocalization should be considered in developing a mouse model for humanPanK2 deficiency.

Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy

NASA Astrophysics Data System (ADS)

Forman, C. J.; Wang, N.; Yang, Z. Y.; Mowat, C. G.; Jarvis, S.; Durkan, C.; Barker, P. D.

2013-05-01

Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.

Decreased cathepsin K levels in human atherosclerotic plaques are associated with plaque instability.

PubMed

Zhao, Huiying; Qin, Xiujiao; Wang, Shuai; Sun, Xiwei; Dong, Bin

2017-10-01

Investigating the determinants and dynamics of atherosclerotic plaque instability is a key area of current cardiovascular research. Extracellular matrix degradation from excessive proteolysis induced by enzymes such as cathepsin K (Cat K) is implicated in the pathogenesis of unstable plaques. The current study assessed the expression of Cat K in human unstable atherosclerotic plaques. Specimens of popliteal arteries with atherosclerotic plaques were classified as stable (<40% lipid core plaque area; n=6) or unstable (≥40% lipid core plaque area; n=14) based on histopathological examinations of hematoxylin and eosin stained sections. The expression of Cat K and cystatin C (Cys C) were assessed by immunohistochemical examination and levels of Cat K mRNA were detected by semi-quantitative reverse transcriptase polymerase chain reaction. Morphological changes including a larger lipid core, endothelial proliferation with foam cells and destruction of internal elastic lamina were observed in unstable atherosclerotic plaques. In unstable plaques, the expression of Cat K protein and mRNA was upregulated, whereas Cys C protein expression was downregulated. The interplay between Cat K and Cys C may underlie the progression of plaques from stable to unstable and the current study indicated that Cat K and Cys C are potential targets for preventing and treating vulnerable atherosclerotic plaque ruptures.

Detecting T-cell reactivity to whole cell vaccines

PubMed Central

Brusic, Ana; Hainz, Ursula; Wadleigh, Martha; Neuberg, Donna; Su, Mei; Canning, Christine M.; DeAngelo, Daniel J.; Stone, Richard M.; Lee, Jeng-Shin; Mulligan, Richard C.; Ritz, Jerome; Dranoff, Glenn; Sasada, Tetsuro; Wu, Catherine J.

2012-01-01

BCR-ABL+ K562 cells hold clinical promise as a component of cancer vaccines, either as bystander cells genetically modified to express immunostimulatory molecules, or as a source of leukemia antigens. To develop a method for detecting T-cell reactivity against K562 cell-derived antigens in patients, we exploited the dendritic cell (DC)-mediated cross-presentation of proteins generated from apoptotic cells. We used UVB irradiation to consistently induce apoptosis of K562 cells, which were then fed to autologous DCs. These DCs were used to both stimulate and detect antigen-specific CD8+ T-cell reactivity. As proof-of-concept, we used cross-presented apoptotic influenza matrix protein-expressing K562 cells to elicit reactivity from matrix protein-reactive T cells. Likewise, we used this assay to detect increased anti-CML antigen T-cell reactivity in CML patients that attained long-lasting clinical remissions following immunotherapy (donor lymphocyte infusion), as well as in 2 of 3 CML patients vaccinated with lethally irradiated K562 cells that were modified to secrete high levels of granulocyte macrophage colony-stimulating factor (GM-CSF). This methodology can be readily adapted to examine the effects of other whole tumor cell-based vaccines, a scenario in which the precise tumor antigens that stimulate immune responses are unknown. PMID:23170257

Compound K induced apoptosis via endoplasmic reticulum Ca2+ release through ryanodine receptor in human lung cancer cells.

PubMed

Shin, Dong-Hyun; Leem, Dong-Gyu; Shin, Ji-Sun; Kim, Joo-Il; Kim, Kyung-Tack; Choi, Sang Yoon; Lee, Myung-Hee; Choi, Jung-Hye; Lee, Kyung-Tae

2018-04-01

Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Compound K-induced ER stress was confirmed through increased phosphorylation of eIF2α and protein levels of GRP78/BiP, XBP-1S, and IRE1α in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular Ca 2+ chelator) or dantrolene (an RyR channel antagonist). These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-induced apoptosis. Cell survival and intracellular Ca 2+ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway.

Deregulated expression of Cdc6 as BCR/ABL-dependent survival factor in chronic myeloid leukemia cells.

PubMed

Zhang, Jia-Hua; He, Yan-Li; Zhu, Rui; Du, Wen; Xiao, Jun-Hua

2017-06-01

Chronic myeloid leukemia is characterized by the presence of the reciprocal translocation t(9;22) and the BCR/ABL oncogene. The BCR/ABL oncogene activates multiple signaling pathways and involves the dysregulation of oncogenes during the progression of chronic myeloid leukemia. The cell division cycle protein 6, an essential regulator of DNA replication, is elevated in some human cancer cells. However, the expression of cell division cycle protein 6 in chronic myeloid leukemia and the underlying regulatory mechanism remain to be elucidated. In this study, our data showed that cell division cycle protein 6 expression was significantly upregulated in primary chronic myeloid leukemia cells and the chronic myeloid leukemia cell line K562 cells, as compared to the normal bone marrow mononuclear cells. BCR/ABL kinase inhibitor STI571 or BCR/ABL small interfering RNA could significantly downregulate cell division cycle protein 6 messenger RNA expression in K562 cells. Moreover, phosphoinositide 3-kinase/AKT pathway inhibitor LY294002 and Janus kinase/signal transducer and activator of transcription pathway inhibitor AG490 could downregulate cell division cycle protein 6 expression in K562 cells, but not RAS/mitogen-activated protein kinase pathway inhibitor PD98059 had such effect. Cell division cycle protein 6 gene silencing by small interfering RNA effectively resulted in decrease of proliferation, increase of apoptosis, and arrest of cell cycle in K562 cells. These findings have demonstrated that cell division cycle protein 6 overexpression may contribute to the high proliferation and low apoptosis in chronic myeloid leukemia cells and can be regulated by BCR/ABL signal transduction through downstream phosphoinositide 3-kinase/Akt and Janus kinase/signal transducer and activator of transcription pathways, suggesting cell division cycle protein 6 as a potential therapeutic target in chronic myeloid leukemia.

Anticancer effects of Bilberry anthocyanins compared with NutraNanoSphere encapsulated Bilberry anthocyanins.

PubMed

Thibado, Seth P; Thornthwaite, Jerry T; Ballard, Thomas K; Goodman, Brandon T

2018-02-01

Rapidly accumulating laboratory and clinical research evidence indicates that anthocyanins exhibit anticancer activity and the evaluation of bilberry anthocyanins as chemo-preventive agents is progressing. It has previously been demonstrated that anthocyanins upregulate tumor suppressor genes, induce apoptosis in cancer cells, repair and protect genomic DNA integrity, which is important in reducing age-associated oxidative stress, and improve neuronal and cognitive brain function. Bilberry anthocyanins have pronounced health effects, even though they have a low bioavailability. To increase the bioavailability, Bilberry was encapsulated in 5.5 nm diameter liposomal micelles, called NutraNanoSpheres (NNS), at a concentration of 2.5 mg/50 µl [25% (w/w) anthocyanins]. These Bilberry NNS were used to study the apoptotic/cytotoxic effects on K562 Human Erythroleukemic cancer cells. Flow cytometric fluorescent quantification of the uptake of propidium iodide in a special cell viability formulation into dead K562 cells was used to determine the effects of Bilberry on the viability of K562 cells. The concentrations of Bilberry that demonstrated the greatest levels of percentage inhibition, relative to the control populations, were biphasic, revealing a 60-70% inhibition between 0.018-1.14 mg/ml (n=6) and 60% inhibition at 4 mg/ml. The lowest percentage inhibition (30%) occurred at 2 mg/ml. The lethal dose 50 was determined to be 0.01-0.04 mg/ml of Bilberry per 105 K562 cells at 72 h of cell culture exposure. At 48 h incubation, the highest percentage of inhibition was only 27%, suggesting involvement of a long-term apoptotic event. These levels, which demonstrated direct cytotoxic effects, were 8-40 times lower than levels required for Bilberry that is not encapsulated. The increase in bioavailability with the Bilberry NNS and its water solubility demonstrated the feasibility of using Bilberry NNS in cancer patient clinical trials.

Anticancer effects of Bilberry anthocyanins compared with NutraNanoSphere encapsulated Bilberry anthocyanins

PubMed Central

Thibado, Seth P.; Thornthwaite, Jerry T.; Ballard, Thomas K.; Goodman, Brandon T.

2018-01-01

Rapidly accumulating laboratory and clinical research evidence indicates that anthocyanins exhibit anticancer activity and the evaluation of bilberry anthocyanins as chemo-preventive agents is progressing. It has previously been demonstrated that anthocyanins upregulate tumor suppressor genes, induce apoptosis in cancer cells, repair and protect genomic DNA integrity, which is important in reducing age-associated oxidative stress, and improve neuronal and cognitive brain function. Bilberry anthocyanins have pronounced health effects, even though they have a low bioavailability. To increase the bioavailability, Bilberry was encapsulated in 5.5 nm diameter liposomal micelles, called NutraNanoSpheres (NNS), at a concentration of 2.5 mg/50 µl [25% (w/w) anthocyanins]. These Bilberry NNS were used to study the apoptotic/cytotoxic effects on K562 Human Erythroleukemic cancer cells. Flow cytometric fluorescent quantification of the uptake of propidium iodide in a special cell viability formulation into dead K562 cells was used to determine the effects of Bilberry on the viability of K562 cells. The concentrations of Bilberry that demonstrated the greatest levels of percentage inhibition, relative to the control populations, were biphasic, revealing a 60–70% inhibition between 0.018–1.14 mg/ml (n=6) and 60% inhibition at 4 mg/ml. The lowest percentage inhibition (30%) occurred at 2 mg/ml. The lethal dose 50 was determined to be 0.01–0.04 mg/ml of Bilberry per 105 K562 cells at 72 h of cell culture exposure. At 48 h incubation, the highest percentage of inhibition was only 27%, suggesting involvement of a long-term apoptotic event. These levels, which demonstrated direct cytotoxic effects, were 8–40 times lower than levels required for Bilberry that is not encapsulated. The increase in bioavailability with the Bilberry NNS and its water solubility demonstrated the feasibility of using Bilberry NNS in cancer patient clinical trials. PMID:29399357

Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.

2012-03-30

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expressionmore » of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.« less

Elemental maps in human allantochorial placental vessels cells: 1. High K + and acetylcholine effects

NASA Astrophysics Data System (ADS)

Michelet-Habchi, C.; Barberet, Ph.; Dutta, R. K.; Guiet-Bara, A.; Bara, M.; Moretto, Ph.

2003-09-01

Regulation of vascular tone in the fetal extracorporeal circulation most likely depends on circulating hormones, local paracrine mechanisms and changes in membrane potential of vascular smooth muscle cells (VSMCs) and of vascular endothelial cells (VECs). The membrane potential is a function of the physiological activities of ionic channels (particularly, K + and Ca 2+ channels in these cells). These channels regulate the ionic distribution into these cells. Micro-particle induced X-ray emission (PIXE) analysis was applied to determine the ionic composition of VSMC and of VEC in the placental human allantochorial vessels in a physiological survival medium (Hanks' solution) modified by the addition of acetylcholine (ACh: which opens the calcium-sensitive K + channels, K Ca) and of high concentration of K + (which blocks the voltage-sensitive K + channels, K df). In VSMC (media layer), the addition of ACh induced no modification of the Na, K, Cl, P, S, Mg and Ca concentrations and high K + medium increased significantly the Cl and K concentrations, the other ion concentrations remaining constant. In endothelium (VEC), ACh addition implicated a significant increase of Na and K concentration, and high K + medium, a significant increase in Cl and K concentration. These results indicated the importance of K df, K Ca and K ATP channels in the regulation of K + intracellular distribution in VSMC and VEC and the possible intervention of a Na-K-2Cl cotransport and corroborated the previous electrophysiological data.

Implementation of a SPR immunosensor for the simultaneous detection of the 22K and 20K hGH isoforms in human serum samples.

PubMed

de Juan-Franco, Elena; Rodríguez-Frade, J M; Mellado, M; Lechuga, Laura M

2013-09-30

We have implemented a Surface Plasmon Resonance (SPR) immunosensor based on a sandwich assay for the simultaneous detection of the two main hGH isoforms, of 22 kDa (22K) and 20 kDa (20K). An oriented-antibody sensor surface specific for both hormone isoforms was assembled by using the biotin-streptavidin system. The immunosensor functionality was checked for the direct detection of the 22K hGH isoform in buffer, which gave high specificity and reproducibility (intra and inter-assay mean coefficients of variation of 8.23% and 9% respectively). The selective determination of the 22K and 20K hGH isoforms in human serum samples in a single assay was possible by using two specific anti-hGH monoclonal antibodies. The detection limit for both hormone isoforms was 0.9 ng mL(-1) and the mean coefficient of variation was below 7.2%. The excellent reproducibility and sensitivity obtained indicate the high performance of this immunosensor for implementing an anti-doping test. Copyright © 2013 Elsevier B.V. All rights reserved.

[Apoptosis of human leukemic cells induced by topoisomerase I and II inhibitors].

PubMed

Solary, E; Dubrez, L; Eymin, B; Bertrand, R; Pommier, Y

1996-03-01

Comparison between five human leukemic lines (BV173, HL60, U937, K562, KCL22) suggest that the main determinant of their sensitivity to topoisomerase I (camptothecin) and II (VP-16) inhibitors is their ability to regulate cell cycle progression in response to specific DNA damage, then to die through apoptosis: the more the cells inhibit cell cycle progression, the less sensitive they are. The final pathway of apoptosis induction involves a cytoplasmic signal, active at neutral pH, needing magnesium, sensitive to various protease inhibitors and activated directly by staurosporine. Modulators of intracellular signaling (calcium chelators, calmodulin inhibitors, PKC modulators, kinase and phosphatase inhibitors) have no significant influence upon apoptosis induction. Conversely, apoptosis induction pathway is modified during monocytic differentiation of HL60 cells induced by phorbol esters. Lastly, poly(ADP-ribosyl)ation and chromatine structure should regulate apoptotic DNA fragmentation that is prevented by 3-aminobenzamide and spermine, respectively.

Synthesis of calbindin-D28K during mineralization in human bone marrow stromal cells.

PubMed Central

Faucheux, C; Bareille, R; Amedee, J

1998-01-01

1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to modulate Ca2+ metabolism in several cell types. Vitamin-D-dependent calcium binding proteins such as calbindin-D28K (28 kDa calcium binding proteins) have been shown to be regulated by 1,25(OH)2D3 but the mechanisms controlling calbindin synthesis are still poorly understood in human osteoblast cell culture models. The human bone marrow stromal cells (HBMSC) described in this paper developed a calcified matrix, expressed osteocalcin (OC), osteopontin (OP) and responded to 1,25(OH)2D3. The expression of vitamin D receptor mRNA was demonstrated by reverse transcription-PCR. Calbindin-D28K protein was identified only in cells arising from the sixth subculture, which exhibited a calcified matrix and all of the osteoblastic markers, e.g. OC and OP. It was demonstrated by dot-immunodetection using immunological probes, and by in situ hybridization using labelled cDNA probes. Moreover, vitamin D3 enhanced calbindin-D28K synthesis as well as OC synthesis and alkaline phosphatase activity. Uptake of 45Ca induced into the matrix by 1,25(OH)2D3 supports the hypothesis that the calcium-enriched matrix could trap calbindin-D proteins. In conclusion, the studies in vitro described in the present paper indicate, for the first time, a possible role of calbindin-D28K in mineralized matrix formation in HBMSC. PMID:9677345

Effect of dexamethasone on skeletal muscle Na+,K+ pump subunit specific expression and K+ homeostasis during exercise in humans

PubMed Central

Nordsborg, Nikolai; Ovesen, Jakob; Thomassen, Martin; Zangenberg, Mathias; Jøns, Christian; Iaia, F Marcello; Nielsen, Jens Jung; Bangsbo, Jens

2008-01-01

The effect of dexamethasone on Na+,K+ pump subunit expression and muscle exchange of K+ during exercise in humans was investigated. Nine healthy male subjects completed a randomized double blind placebo controlled protocol, with ingestion of dexamethasone (Dex: 2 × 2 mg per day) or placebo (Pla) for 5 days. Na+,K+ pump catalytic α1 and α2 subunit expression was ∼17% higher (P < 0.05) and the structural β1 and β2 subunit expression was ∼6–8% higher (P < 0.05) after Dex compared with Pla. During one-legged knee-extension for 10 min at low intensity (LI; 18.6 ± 1.0 W), two moderate intensity (51.7 ± 2.4 W) exercise bouts (MI1: 5 min; 2 min recovery; MI2: exhaustive) and two high-intensity (71.7 ± 2.5 W) exercise bouts (HI1: 1 min 40 s; 2 min recovery; HI2: exhaustive), femoral venous K+ was lower (P < 0.05) in Dex compared with Pla. Thigh K+ release was lower (P < 0.05) in Dex compared with Pla in LI and MI, but not in HI. Time to exhaustion in MI2 tended to improve (393 ± 50 s versus 294 ± 41 s; P = 0.07) in Dex compared with Pla, whereas no difference was detected in HI2 (106 ± 10 s versus 108 ± 9 s). The results indicate that an increased Na+,K+ pump expression per se is of importance for thigh K+ reuptake at the onset of low and moderate intensity exercise, but less important during high intensity exercise. PMID:18174214

In-vitro singlet oxygen threshold dose at PDT with Radachlorin photosensitizer

NASA Astrophysics Data System (ADS)

Klimenko, V. V.; Shmakov, S. V.; Kaydanov, N. E.; Knyazev, N. A.; Kazakov, N. V.; Rusanov, A. A.; Bogdanov, A. A.; Dubina, M. V.

2017-07-01

In this present study we investigate the Radachlorin photosensitizer accumulation in K562 cells and Hela cells and determined the cell viability after PDT. Using the macroscopic singlet oxygen modeling and cellular photosensitizer concentration the singlet oxygen threshold doses for K562 cells and Hela cells were calculated.

Part-1: Design, synthesis and biological evaluation of novel bromo-pyrimidine analogs as tyrosine kinase inhibitors.

PubMed

Munikrishnappa, Chandrashekar Suradhenupura; Puranik, Sangamesh B; Kumar, G V Suresh; Prasad, Y Rajendra

2016-08-25

A novel series of 5-bromo-pyrimidine derivatives (5a-l, 6a-h, 9a-m and 10a-d) were synthesized through multi step reactions starting from 5-bromo-2,4-dichloro pyrimidine. The newly synthesized compounds were characterized using elemental analysis and spectral data (IR, (1)H NMR, (13)C NMR and LC-MS) analysis. The titled compounds were evaluated for their in vitro cytotoxic activity against tumor cell lines panel consisted of HCT116 (human colon cancer cell line), A549 (human lung cancer cell line), K562 (human chronic myeloid leukemia cell line), U937 (human acute monocytic myeloid leukemia cell line), and L02 (human normal cell line) by using MTT assay Mosmann's method. As most of the compounds are highly potent against K562 cells, all the synthesized compounds were evaluated for Bcr/Abl tyrosine kinase inhibitory activity by using well-established ADP-Glo assay method. Dasatinib was utilized as positive control to validate in both biological evaluations. The biological activity revealed that the compounds 5c, 5e, 6g, 9e, 9f and 10c were potent Bcr/Abl kinase inhibitors among the titled compounds. Thus these compounds may be promising lead compounds to be developed as an alternative for current Dasatinib therapy. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

By activating Fas/ceramide synthase 6/p38 kinase in lipid rafts, Stichoposide D inhibits growth of leukemia xenografts

PubMed Central

Yun, Seong-Hoon; Park, Eun-Seon; Shin, Sung-Won; Ju, Mi-Ha; Han, Jin-Yeong; Jeong, Jin-Sook; Kim, Sung-Hyun; Stonik, Valentin A.; Kwak, Jong-Young; Park, Joo-In

2015-01-01

Stichoposide D (STD) is a marine triterpene glycoside isolated from sea cucumbers. We examined the molecular mechanisms underlying the antitumor activity of STD in human leukemia cells. The role of Fas (CD95), ceramide synthase 6 (CerS6) and p38 kinase during STD-induced apoptosis was examined in human leukemia cells. In addition, the antitumor effects of STD in K562 and HL-60 leukemia xenograft models were investigated. We found that STD induces Fas translocation to lipid rafts, and thus mediates cell apoptosis. We also observed the activation of CerS6 and p38 kinase during STD-induced apoptosis. The use of methyl-β-cyclodextrin and nystatin to disrupt lipid rafts prevents the clustering of Fas and the activation of CerS6 and p38 kinase, and also inhibits STD-induced apoptosis. Specific inhibition by Fas, CerS6, and p38 kinase siRNA transfection partially blocked STD-induced apoptosis. In addition, STD has antitumor activity through the activation of CerS6 and p38 kinase without displaying any toxicity in HL-60 and K562 xenograft models. We observed that the anti-tumor effect of STD is partially prevented in CerS6 shRNA-silenced xenograft models. We first report that Fas/CerS6/p38 kinase activation in lipid rafts by STD is involved in its anti-leukemic activity. We also established that STD is able to enhance the chemosensitivity of K562 cells to etoposide or Ara-C. These data suggest that STD may be used alone or in combination with other chemotherapeutic agents to treat leukemia. PMID:26318294

Polychromatic Light (480-3400 nm) Upregulates Sensitivity of Tumor Cells to Lysis by Natural Killers.

PubMed

Knyazev, Nickolay A; Samoilova, Kira A; Abrahamse, Heidi; Filatova, Natalia A

2016-09-01

This study evaluates the participation of immunological mechanisms of downregulation of murine hepatoma cells MH22a after direct exposure to polychromatic polarized light. Previous studies have shown that exposure to a combination of visible (VIS) and infrared (IR) light leads to decreased tumorigenicity of the murine hepatoma cells MH22a, which correlated with an increase in the amount of cells with reorganized cytoskeleton in the submembrane region. The mechanism of tumor inhibition and elimination has not been determined. Polychromatic light (480-3400 nm) has been used at doses of 4.8 and 9.6 J/cm(2) to determine the sensitivity of murine MH22a cells and human erythroleukemia cells K562 exposed to this light, to lysis by effector cells of innate immunity (NK cells), and enhancement of the glycocalyx of the studied tumor cells. This was determined using flow cytometry, the H(3)-uridine cytotoxic test followed by spectrophotometry. VIS-IR light increases the sensitivity of MH-22a cells at a dose 4.8 J/cm(2) and K562 cells at 9.6 J/cm(2). The enhancement of sensitivity of tumor cells to NK lysis changed their ability to absorb alcian blue, reflecting a change in the expression of the glycocalyx. Increasing the sensitivity of the murine tumor cells MH22a and human K562 irradiated VIS-IR light correlated with a change in the expression of their glycocalyx. The results of the present study demonstrate that the reduction of tumorigenicity of irradiated tumor cells is due to their sensitivity to lysis by NK cells of the immune system.

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

PubMed

Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

2018-05-15

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret

The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

NASA Astrophysics Data System (ADS)

Wu, Sangwook

2016-04-01

The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

Tissue-specific expression of the gene coding for human Clara cell 10-kD protein, a phospholipase A2-inhibitory protein.

PubMed Central

Peri, A; Cordella-Miele, E; Miele, L; Mukherjee, A B

1993-01-01

Clara cell 10-kD protein (cc10kD), a secretory phospholipase A2 inhibitor, is suggested to be the human counterpart of rabbit uteroglobin (UG). Because cc10kD is expressed constitutively at a very high level in the human respiratory epithelium, the 5' region of its gene may be useful in achieving organ-specific expression of recombinant DNA in gene therapy of diseases such as cystic fibrosis. However, it is important to establish the tissue-specific expression of this gene before designing gene transfer experiments. Since the UG gene in the rabbit is expressed in many other organs besides the lung and the endometrium, we investigated the organ and tissue specificity of human cc10kD gene expression using polymerase chain reaction, nucleotide sequence analysis, immunofluorescence, and Northern blotting. Our results indicate that, in addition to the lung, cc10kD is expressed in several nonrespiratory organs, with a distribution pattern very similar, if not identical, to that of UG in the rabbit. These results underscore the necessity for more detailed analyses of the 5' region of the human cc10kD gene before its usefulness in gene therapy could be fully assessed. These data also suggest that cc10kD and UG may have similar physiological function(s). Images PMID:8227325

In situ Localization of the Human Multidrug‐resistance Gene mRNA Using Thymine‐Thymine Dimerized Single‐stranded cDNA

PubMed Central

Koji, Takehiko; Ueda, Kazumitsu; Pastan, Ira; Gottesman, Michael M.; Nakane, Paul K.; Mori, Shigeo

1990-01-01

In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T‐T dimerized single‐stranded DNA (300–400 bases) so that they could penetrate well into the cytoplasm. The hybridized T‐T DNA was detected immunohisto‐chemically using rabbit anti‐T‐T DNA antibody (Ab) and peroxidase‐labeled goat anti‐rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug‐resistant cell lines K562/ADM, CEM/VLB, 2780ad, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene. PMID:1977730

Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors

PubMed Central

Rycaj, Kiera; Plummer, Joshua B.; Li, Ming; Yin, Bingnan; Frerich, Katherine; Garza, Jeremy G.; Shen, Jianjun; Lin, Kevin; Yan, Peisha; Glynn, Sharon A.; Dorsey, Tiffany H.; Hunt, Kelly K.; Ambs, Stefan; Johanning, Gary L.

2012-01-01

Background The envelope (env) protein of the human endogenous retrovirus type K (HERV-K) family is commonly expressed on the surface of breast cancer cells. We assessed whether HERV-K env is a potential target for antibody-based immunotherapy of breast cancer. Methods We examined the expression of HERV-K env protein in various malignant (MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1) and nonmalignant (MCF-10A and MCF-10AT) human breast cell lines by immunoblot, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry. Anti-HERV-K env monoclonal antibodies (mAbs; 6H5, 4D1, 4E11, 6E11, and 4E6) were used to target expression of HERV-K, and antitumor effects were assessed by quantifying growth and apoptosis of breast cancer cells in vitro, and tumor growth in vivo in mice (n = 5 per group) bearing xenograft tumors. The mechanisms responsible for 6H5 mAb–mediated effects were investigated by microarray assays, flow cytometry, immunoblot, and immunofluorescence staining. The expression of HERV-K env protein was assessed in primary breast tumors (n = 223) by immunohistochemistry. All statistical tests were two-sided. Results The expression of HERV-K env protein in malignant breast cancer cell lines was substantially higher than nonmalignant breast cells. Anti–HERV-K-specific mAbs inhibited growth and induced apoptosis of breast cancer cells in vitro. Mice treated with 6H5 mAb showed statistically significantly reduced growth of xenograft tumors compared with mice treated with control immunoglobulin (control [mIgG] vs 6H5 mAb, for tumors originating from MDA-MB-231 cells, mean size = 1448.33 vs 475.44 mm3; difference = 972.89 mm3, 95% CI = 470.17 to 1475.61 mm3; P < .001). Several proteins involved in the apoptotic signaling pathways were overexpressed in vitro in 6H5 mAb–treated malignant breast cells compared with mIgG-treated control. HERV-K expression was detected in 148 (66%) of 223 primary breast tumors, and a higher rate of

A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

PubMed

Abraham, S; Solomon, W B

2000-09-19

We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin

Poultry as reservoir for extraintestinal pathogenic Escherichia coli O45:K1:H7-B2-ST95 in humans.

PubMed

Mora, Azucena; Viso, Susana; López, Cecilia; Alonso, María Pilar; García-Garrote, Fernando; Dabhi, Ghizlane; Mamani, Rosalía; Herrera, Alexandra; Marzoa, Juan; Blanco, Miguel; Blanco, Jesús E; Moulin-Schouleur, Maryvonne; Schouler, Catherine; Blanco, Jorge

2013-12-27

Escherichia coli strains O45:K1:H7 are implicated in severe human infections such as meningitis. Since an increasing prevalence of serogroup O45 among avian pathogenic (APEC) and human extraintestinal pathogenic (ExPEC) E. coli strains isolated in Spain have been noticed, the aims of the present study were to investigate similarities between poultry and human O45 isolates, and to investigate the evolutionary relationship of ST95 types. The genetic relatedness and virulence gene profiles of 55 O45 APEC obtained from an avian colibacillosis collection (1991-2011) and 19 human O45 ExPEC from a human septicemic/uropathogenic (UPEC) E. coli collection (1989-2010) were determined by multilocus sequence typing (MLST), pulsed-field-gel-electrophoresis (PFGE), ECOR phylogrouping, and PCR-based genotyping. Two main clonal groups were established. The most prevalent and highly pathogenic O45:K1:H7-B2-ST95 shows a successful persistence since the 90s to the present, with parallel evolution both in human and poultry, on the basis of their PFGE and virulence gene profile similarities (9 human strains and 15 avian strains showed ≥85% PFGE identity). Comparison of this group with other ST95 closely related members (O1:K1:H7 and O18:K1:H7 isolates from our collections) shows pathogenic specialization through conserved virulence genotypes. The other prevalent O45 clonal group characterized in this study, the O45:HNM/H19-D-ST371/ST2676 was only detected in APEC strains suggesting host specificity. In conclusion, poultry could be acting as a reservoir of O45:K1:H7-B2-ST95 and other pathogenic ST95 serotypes in humans. Further studies would be necessary to clarify if pathogenic mechanisms used by ST95 strains are the same in avian and human hosts. Copyright © 2013 Elsevier B.V. All rights reserved.

Dissecting the mechanism of histone deacetylase inhibitors to enhance the activity of zinc finger nucleases delivered by integrase-defective lentiviral vectors.

PubMed

Joglekar, Alok V; Stein, Libby; Ho, Michelle; Hoban, Megan D; Hollis, Roger P; Kohn, Donald B

2014-07-01

Integrase-defective lentiviral vectors (IDLVs) have been of limited success in the delivery of zinc finger nucleases (ZFNs) to human cells, due to low expression. A reason for reduced gene expression has been proposed to involve the epigenetic silencing of vector genomes, carried out primarily by histone deacetylases (HDACs). In this study, we tested valproic acid (VPA), a known HDAC inhibitor (HDACi), for its ability to increase transgene expression from IDLVs, especially in the context of ZFN delivery. Using ZFNs targeting the human adenosine deaminase (ADA) gene in K562 cells, we demonstrated that treatment with VPA enhanced ZFN expression by up to 3-fold, resulting in improved allelic disruption at the ADA locus. Furthermore, three other U.S. Food and Drug Administration-approved HDACis (vorinostat, givinostat, and trichostatin-A) exhibited a similar effect on the activity of ZFN-IDLVs in K562 cells. In primary human CD34(+) cells, VPA- and vorinostat-treated cells showed higher levels of expression of both green fluorescent protein (GFP) as well as ZFNs from IDLVs. A major mechanism for the effects of HDAC inhibitors on improving expression was from their modulation of the cell cycle, and the influence of heterochromatinization was determined to be a lesser contributing factor.

Escherichia coli K1 induces IL-8 expression in human brain microvascular endothelial cells.

PubMed

Galanakis, Emmanouil; Di Cello, Francescopaolo; Paul-Satyaseela, Maneesh; Kim, Kwang Sik

2006-12-01

Microbial penetration of the blood-brain barrier (BBB) into the central nervous system is essential for the development of meningitis. Considerable progress has been achieved in understanding the pathophysiology of meningitis, however, relatively little is known about the early inflammatory events occurring at the time of bacterial crossing of the BBB. We investigated, using real-time quantitative PCR, the expression of the neutrophil chemoattractants alpha-chemokines CXCL1 (Groalpha) and CXCL8 (IL-8), and of the monocyte chemoattractant beta-chemokine CCL2 (MCP-1) by human brain microvascular endothelial cells (HBMEC) in response to the meningitis-causing E. coli K1 strain RS218 or its isogenic mutants lacking the ability to bind to and invade HBMEC. A nonpathogenic, laboratory E. coli strain HB101 was used as a negative control. CXCL8 was shown to be significantly expressed in HBMEC 4 hours after infection with E. coli K1, while no significant alterations were noted for CXCL1 and CCL2 expression. This upregulation of CXCL8 was induced by E. coli K1 strain RS218 and its derivatives lacking the ability to bind and invade HBMEC, but was not induced by the laboratory strain HB101. In contrast, no upregulation of CXCL8 was observed in human umbilical vein endothelial cells (HUVEC) after stimulation with E. coli RS218. These findings indicate that the CXCL8 expression is the result of the specific response of HBMEC to meningitis-causing E. coli K1.

Human mass balance, metabolite profile and identification of metabolic enzymes of [¹⁴C]ASP015K, a novel oral janus kinase inhibitor.

PubMed

Oda, Kazuo; Cao, Ying J; Sawamoto, Taiji; Nakada, Naoyuki; Fisniku, Ogert; Nagasaka, Yasuhisa; Sohda, Kin-Ya

2015-01-01

1. The human mass balance of (14)C-labelled ASP015K ([(14)C]ASP015K), an orally bioavailable Janus kinase (JAK) inhibitor, was characterized in six healthy male subjects after a single oral dose of [(14)C]ASP015K (100 mg, 3.7 MBq) in solution. [(14)C]ASP015K was rapidly absorbed with tmax of 1.6 and 1.8 h for ASP015K and total radioactivity in plasma, respectively. Mean recovery in urine and feces amounted to 36.8% and 56.6% of the administered dose, respectively. The main components of radioactivity in plasma and urine were ASP015K and M2 (5'-O-sulfo ASP015K). In feces, ASP015K and M4 (7-N-methyl ASP015K) were the main components. 2. In vitro study of ASP015K metabolism showed that the major isozyme contributing to the formation of M2 was human sulfotransferase (SULT) 2A1 and of M4 was nicotinamide N-methyltransferase (NNMT). 3. The in vitro intrinsic clearance (CLint_in vitro) of M4 formation from ASP015K in human liver cytosol (HLC) was 11-fold higher than that of M2. The competitive inhibitory effect of nicotinamide on M4 formation in the human liver was considered the reason for high CLint_in vitro of M4 formation, while each metabolic pathway made a near equal contribution to the in vivo elimination of ASP015K. ASP015K was cleared by multiple mechanisms.

Down-regulation of human endogenous retrovirus type K (HERV-K) viral env RNA in pancreatic cancer cells decreases cell proliferation and tumor growth

PubMed Central

Li, Ming; Radvanyi, Laszlo; Yin, Bingnan; Li, Jia; Chivukula, Raghavender; Lin, Kevin; Lu, Yue; Shen, JianJun; Chang, David Z.; Li, Donghui; Johanning, Gary L.; Wang-Johanning, Feng

2017-01-01

Purpose We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer (PC). Experimental Design shRNA was employed to knockdown (KD) the expression of HERV-K in PC cells. Results HERV-K env expression was detected in seven PC cell lines and in 80% of PC patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several PC cell lines. RT activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in PC patient sera (N=106) than in normal donor sera (N=40). Importantly, the in vitro and in vivo growth rates of three PC cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several PC cells or tumors. Conclusion These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in PC. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of PC, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis and immunotherapy of PC. PMID:28679769

Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

PubMed Central

Kim, Dong-Hyun; Kim, Baek

2011-01-01

The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors. PMID:21765914

CCQM K6.2 determination of total cholesterol in human serum

NASA Astrophysics Data System (ADS)

Wise, Stephen A.; Phinney, Karen W.; Duewer, David L.; Sniegoski, Lorna T.; Welch, Michael J.; Pabello, Guiomar; Avila Caldero, Marco A.; Qinde, Liu; Kooi, Lee Tong; Rego, Eliane; Garrido, Bruno; Allegri, Gabriella; de La Cruz, Marcia; Barrabin, Juliana; Puglisi, Celia; Lopez, Eduardo; Lee, Hwashim; Kim, Byungjoo; Delatour, Vincent; Heuillet, Maud; Nammoonnoy, Jintana; Ceyhan Gören, Ahmet; Bilsel, Gokhan; Konopelko, L.; Krylov, A.; Lopushanskaya, E.

2018-01-01

Cholesterol is one of the most frequently measured substances in human blood/serum to assist in assessing the health status of individuals. Because of its clinical significance, CCQM-K6 determination of cholesterol in serum was completed in 2000 as one of the first key comparison (KC) studies performed within the Organic Analysis Working Group (OAWG). The first subsequent KC for cholesterol, CCQM-K6.1, was completed in 2001. Measurements for this second subsequent, CCQM-K6.2, were completed in 2012. These subsequent comparisons were conducted to enable CCQM members that had not participated in earlier studies to demonstrate their capabilities to measure a nonpolar (pKow < ‑2), low molecular mass (100 g/mol to 500 g/mol) metabolite in human serum at relatively high concentrations (1 mg/g to 3 mg/g) found in normal populations. Successful participation in CCQM-K6.2 demonstrated capabilities in analysis of complex biological matrices including sample preparation (extraction, derivatization), LC or GC separation, and quantification using an isotope dilution mass spectrometry approach. Normally in a subsequent KC, no key comparison reference value (KCRV) would be established and assessment of performance would be via the deviation of participants' results to the anchor institute's results, adjusted to account for the anchor's performance in the original comparison versus its KCRV. Due to the very long-time period since the original key comparison, the OAWG decided that this did not represent the best approach to assess performance in what is a relatively complex measurement. Given the excellent agreement between the anchor institute's results and robust consensus summary of the participants' values, the reference value for this study was taken as the anchor institute's result and treated as a 'KCRV'. Seven of the nine participants demonstrated agreement with the reference value. Main text To reach the main text of this paper, click on Final Report. Note that this text

Nucleosome eviction along with H3K9ac deposition enhances Sox2 binding during human neuroectodermal commitment

PubMed Central

Du, Yanhua; Liu, Zhenping; Cao, Xinkai; Chen, Xiaolong; Chen, Zhenyu; Zhang, Xiaobai; Zhang, Xiaoqing; Jiang, Cizhong

2017-01-01

Neuroectoderm is an important neural precursor. However, chromatin remodeling and its epigenetic regulatory roles during the differentiation of human neuroectodermal cells (hNECs) from human embryonic stem cells (hESCs) remain largely unexplored. Here, we obtained hNECs through directed differentiation from hESCs, and determined chromatin states in the two cell types. Upon differentiation, H2A.Z-mediated nucleosome depletion leads to an open chromatin structure in promoters and upregulates expression of neuroectodermal genes. Increase in H3K9ac signals and decrease in H3K27me3 signals in promoters result in an active chromatin state and activate neuroectodermal genes. Conversely, decrease in H3K9ac signals and increase in H3K27me3 signals in promoters repress pluripotency genes. Moreover, H3K9ac signals facilitate the pluripotency factor Sox2 binding to target sites unique to hNECs. Knockdown of the acetyltransferase Kat2b erases H3K9ac signals, disrupts Sox2 binding, and fails the differentiation. Our results demonstrate a hierarchy of epigenetic regulation of gene expression during the differentiation of hNECs from hESCs through chromatin remodeling. PMID:28475175

Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway.

PubMed

Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Isono, Toshihito; Nakamura, Yuki; Saitoh, Issei; Hayasaki, Haruaki; Yamaguchi, Masaya; Kawabata, Shigetada; Terao, Yutaka

2018-01-01

Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine-threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine-threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Ionophore-A23187-induced cellular cytotoxicity: a cell fragment mediated process.

PubMed Central

Nash, G S; Niedt, G W; MacDermott, R P

1980-01-01

Calcium ionophore A23187 was found to induce human white blood cells to kill human red blood cells. Optimal conditions for ionophore-induced cellular cytotoxicity (IICC) included an 18 h time period, an incubation temperature of 25 degrees, a 25:1 or 50:1 killer:target cell ratio,and a final ionophore concentration of 2 . 5 microgram/ml. WBC or granulocytes which were either frozen and thawed three times or sonicated were capable of mediating IICC. As intact cells, granulocytes (67 . 2% cytotoxicity), monocytes (34 . 8%), B cells (22 . 0%) and Null cells (19 . 3%) were effector cells but T cells (7 . 4%) were not. After fragmenting these cells, all cell types including T cells were able to mediate IICC. When cell lines (K562, Chang, and NCTC) were used as effectors, none would mediate IICC when intact. After freezing and thawing, Chang and NCTC would not mediate IICC, whereas K562 cells did. These studies may be indicative of a calcium-dependent, membrane-localized mechanism in cellular cytotoxic processes, and may provide a useful indicator system for isolation of the enzyme systems involved in cellular cytotoxicity. PMID:6773881

Establishment of nude mice with complete loss of lymphocytes and NK cells and application for in vivo bio-imaging.

PubMed

Kariya, Ryusho; Matsuda, Kouki; Gotoh, Kumiko; Vaeteewoottacharn, Kulthida; Hattori, Shinichiro; Okada, Seiji

2014-01-01

Nude mice are used in human xenograft research; however, only 25-35% of human tumors have been successfully transplanted into nude mice and their application is limited due to high natural killer (NK) cell activity. More severely immunodeficient mice with loss of NK activity are needed to overcome this limitation. Balb/c nude Rag-2(-/-)Jak3(-/-) (Nude-RJ) mice were established by crossing Rag-2(-/-)Jak3(-/-) mice and nude mice. The K562 cell line was implanted subcutaneously to compare tumorigenicity between Nude-RJ mice and Nude mice. The cholangiocarcinoma mCherry expressing cell line (KKU-M213) was implanted subcutaneously, and fluorescence intensity and tumor weight were measured. Nude R/J mice showed complete loss of lymphocytes and NK cells. Xeno-transplantation of K562 cells showed higher proliferation in Nude R/J mice than nude mice. Subcutaneously-transplanted mCherry-transduced KKU-M213 cells were successfully detected with a fluorescence imager. Nude-R/J mice are valuable tools for in vivo imaging studies in biomedical research. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Purpurogemutantin and Purpurogemutantidin, New Drimenyl Cyclohexenone Derivatives Produced by a Mutant Obtained by Diethyl Sulfate Mutagenesis of a Marine-Derived Penicillium purpurogenum G59

PubMed Central

Fang, Shi-Ming; Cui, Cheng-Bin; Li, Chang-Wei; Wu, Chang-Jing; Zhang, Zhi-Jun; Li, Li; Huang, Xiao-Jun; Ye, Wen-Cai

2012-01-01

Two new drimenyl cyclohexenone derivatives, named purpurogemutantin (1) and purpurogemutantidin (2), and the known macrophorin A (3) were isolated from a bioactive mutant BD-1-6 obtained by random diethyl sulfate (DES) mutagenesis of a marine-derived Penicillium purpurogenum G59. Structures and absolute configurations of 1 and 2 were determined by extensive spectroscopic methods, especially 2D NMR and electronic circular dichroism (ECD) analysis. Possible biosynthetic pathways for 1–3 were also proposed and discussed. Compounds 1 and 2 significantly inhibited human cancer K562, HL-60, HeLa, BGC-823 and MCF-7 cells, and compound 3 also inhibited the K562 and HL-60 cells. Both bioassay and chemical analysis (HPLC, LC-ESIMS) demonstrated that the parent strain G59 did not produce 1–3, and that DES-induced mutation(s) in the mutant BD-1-6 activated some silent biosynthetic pathways in the parent strain G59, including one set for 1–3 production. PMID:22822371

Use of Engineered Exosomes Expressing HLA and Costimulatory Molecules to Generate Antigen-specific CD8+ T Cells for Adoptive Cell Therapy.

PubMed

Kim, Sueon; Sohn, Hyun-Jung; Lee, Hyun-Joo; Sohn, Dae-Hee; Hyun, Seung-Joo; Cho, Hyun-Il; Kim, Tai-Gyu

2017-04-01

Dendritic cell-derived exosomes (DEX) comprise an efficient stimulator of T cells. However, the production of sufficient DEX remains a barrier to their broad applicability in immunotherapeutic approaches. In previous studies, genetically engineered K562 have been used to generate artificial antigen presenting cells (AAPC). Here, we isolated exosomes from K562 cells (referred to as CoEX-A2s) engineered to express human leukocyte antigen (HLA)-A2 and costimulatory molecules such as CD80, CD83, and 41BBL. CoEX-A2s were capable of stimulating antigen-specific CD8 T cells both directly and indirectly via CoEX-A2 cross-dressed cells. Notably, CoEX-A2s also generated similar levels of HCMV pp65-specific and MART1-specific CD8 T cells as DEX in vitro. The results suggest that these novel exosomes may provide a crucial reagent for generating antigen-specific CD8 T cells for adoptive cell therapies against viral infection and tumors.

THE HUMAN BEHAVIOR RATING SCALE-BRIEF: A TOOL TO MEASURE 21ST CENTURY SKILLS OF K-12 LEARNERS.

PubMed

Woods-Groves, Suzanne

2015-06-01

Currently there is a call for brief concise measurements to appraise relevant 21st century college readiness skills in K-12 learners. This study employed K-12 teachers' ratings for over 3,000 students for an existing 91-item rating scale, the Human Behavior Rating Scale, that measured the 21st century skills of persistence, curiosity, externalizing affect, internalizing affect, and cognition. Teachers' ratings for K-12 learners were used to develop a brief, concise, and manageable 30-item tool, the Human Behavior Rating Scale-Brief. Results yielded high internal consistency coefficients and inter-item correlations. The items were not biased with regard to student sex or race, and were supported through confirmatory factor analyses. In addition, when teachers' ratings were compared with students' academic and behavioral performance data, moderate to strong relationships were revealed. This study provided an essential first step in the development of a psychometrically sound, manageable, and brief tool to appraise 21st century skills in K-12 learners.

Potent apoptosis-inducing activity of erypoegin K, an isoflavone isolated from Erythrina poeppigiana, against human leukemia HL-60 cells.

PubMed

Hikita, Kiyomi; Hattori, Natsuki; Takeda, Aya; Yamakage, Yuko; Shibata, Rina; Yamada, Saori; Kato, Kuniki; Murata, Tomiyasu; Tanaka, Hitoshi; Kaneda, Norio

2018-01-01

Erypoegin K is an isoflavone isolated from the stem bark of Erythrina poeppigiana. It contains a furan group at the A-ring of the core isoflavone structure and can inhibit the activity of glyoxalase I, an enzyme that catalyzes the detoxification of methylglyoxal (MG), a by-product of glycolysis. In the present study, we found that erypoegin K has a potent cytotoxic effect on human leukemia HL-60 cells. Its cytotoxic effect was much stronger than that of a known glyoxalase I inhibitor S-p-bromobenzylglutathione cyclopentyl diester. Conversely, erypoegin K demonstrated weak cytotoxicity toward normal human peripheral lymphocytes. The treatment of HL-60 cells with erypoegin K significantly induced caspase-3 activity, whereas the pretreatment of the cells with caspase-3 inhibitor suppressed erypoegin K-induced cell death. Furthermore, nuclear condensation and apoptotic genome DNA fragmentation were observed in erypoegin K-treated HL-60 cells. These results indicated that the observed cell death was mediated by apoptosis. In addition, the toxic compound MG was highly accumulated in the culture medium of erypoegin K-treated HL-60 cells, suggesting that cell apoptosis was triggered by extracellular MG. The present study showed that erypoegin K has a potent apoptosis-inducing effect on cancerous cell lines, such as HL-60.

Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

PubMed Central

Majumder, Syamantak; Thieme, Karina; Batchu, Sri N.; Alghamdi, Tamadher A.; Bowskill, Bridgit B.; Kabir, M. Golam; Liu, Youan; Advani, Suzanne L.; White, Kathryn E.; Geldenhuys, Laurette; Tennankore, Karthik K.; Poyah, Penelope; Siddiqi, Ferhan S.

2017-01-01

Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain–containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways. PMID:29227285

Anticancer activity of ferrocenylthiosemicarbazones.

PubMed

Sandra, Cortez-Maya; Elena, Klimova; Marcos, Flores-Alamo; Elena, Martínez-Klimova; Arturo, Ramírez-Ramírez; Teresa, Ramírez Apan; Marcos, Martínez-García

2014-03-01

Aliphatic and aromatic ferrocenylthiosemicarbazones were synthesized. The characterization of the new ferrocenylthiosemicarbazones was done by IR, (1)H-NMR and (13)C-NMR spectroscopy, elemental analysis and X-ray diffraction studies. The biological activity of the obtained compounds was assessed in terms of anticancer activity. Their activity against U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma), K562 (human chronic myelogenous leukemia), HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma) and SKLU-1 (human lung adenocarcinoma) cell lines was studied and compared with cisplatin. All tested compounds showed good activity and the aryl-chloro substituted ferrocenylthiosemicarbazones showed the best anticancer activity.

Characterization of the cloned human intermediate-conductance Ca2+-activated K+ channel.

PubMed

Jensen, B S; Strobaek, D; Christophersen, P; Jorgensen, T D; Hansen, C; Silahtaroglu, A; Olesen, S P; Ahring, P K

1998-09-01

The human intermediate-conductance, Ca2+-activated K+ channel (hIK) was identified by searching the expressed sequence tag database. hIK was found to be identical to two recently cloned K+ channels, hSK4 and hIK1. RNA dot blot analysis showed a widespread tissue expression, with the highest levels in salivary gland, placenta, trachea, and lung. With use of fluorescent in situ hybridization and radiation hybrid mapping, hIK mapped to chromosome 19q13.2 in the same region as the disease Diamond-Blackfan anemia. Stable expression of hIK in HEK-293 cells revealed single Ca2+-activated K+ channels exhibiting weak inward rectification (30 and 11 pS at -100 and +100 mV, respectively). Whole cell recordings showed a noninactivating, inwardly rectifying K+ conductance. Ionic selectivity estimated from bi-ionic reversal potentials gave the permeability (PK/PX) sequence K+ = Rb+ (1.0) > Cs+ (10.4) > Na+, Li+, N-methyl-D-glucamine (>51). NH+4 blocked the channel completely. hIK was blocked by the classical inhibitors of the Gardos channel charybdotoxin (IC50 28 nM) and clotrimazole (IC50 153 nM) as well as by nitrendipine (IC50 27 nM), Stichodactyla toxin (IC50 291 nM), margatoxin (IC50 459 nM), miconazole (IC50 785 nM), econazole (IC50 2.4 microM), and cetiedil (IC50 79 microM). Finally, 1-ethyl-2-benzimidazolinone, an opener of the T84 cell IK channel, activated hIK with an EC50 of 74 microM.

Calcium-activated K(+) channel (K(Ca)3.1) activity during Ca(2+) store depletion and store-operated Ca(2+) entry in human macrophages.

PubMed

Gao, Ya-dong; Hanley, Peter J; Rinné, Susanne; Zuzarte, Marylou; Daut, Jurgen

2010-07-01

STIM1 'senses' decreases in endoplasmic reticular (ER) luminal Ca(2+) and induces store-operated Ca(2+) (SOC) entry through plasma membrane Orai channels. The Ca(2+)/calmodulin-activated K(+) channel K(Ca)3.1 (previously known as SK4) has been implicated as an 'amplifier' of the Ca(2+)-release activated Ca(2+) (CRAC) current, especially in T lymphocytes. We have previously shown that human macrophages express K(Ca)3.1, and here we used the whole-cell patch-clamp technique to investigate the activity of these channels during Ca(2+) store depletion and store-operated Ca(2+) influx. Using RT-PCR, we found that macrophages express the elementary CRAC channel components Orai1 and STIM1, as well as Orai2, Orai3 and STIM2, but not the putatively STIM1-activated channels TRPC1, TRPC3-7 or TRPV6. In whole-cell configuration, a robust Ca(2+)-induced outwardly rectifying K(+) current inhibited by clotrimazole and augmented by DC-EBIO could be detected, consistent with K(Ca)3.1 channel current (also known as intermediate-conductance IK1). Introduction of extracellular Ca(2+) following Ca(2+) store depletion via P2Y(2) receptors induced a robust charybdotoxin (CTX)- and 2-APB-sensitive outward K(+) current and hyperpolarization. We also found that SOC entry induced by thapsigargin treatment induced CTX-sensitive K(+) current in HEK293 cells transiently expressing K(Ca)3.1. Our data suggest that SOC and K(Ca)3.1 channels are tightly coupled, such that a small Ca(2+) influx current induces a much large K(Ca)3.1 channel current and hyperpolarization, providing the necessary electrochemical driving force for prolonged Ca(2+) signaling and store repletion. Copyright 2010 Elsevier Ltd. All rights reserved.

Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny.

PubMed

Georgouli, Mirella; Papadimitriou, Lina; Glymenaki, Maria; Patsaki, Valia; Athanassakis, Irene

2016-06-01

Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

Purification and Characterization of Tagless Recombinant Human Elongation Factor 2 Kinase (eEF-2K) Expressed in Escherichia coli

PubMed Central

Abramczyk, Olga; Tavares, Clint D. J.; Devkota, Ashwini K.; Ryazanov, Alexey G.; Turk, Benjamin E.; Riggs, Austen F.; Ozpolat, Bulent; Dalby, Kevin N.

2012-01-01

The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His6-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ~ 85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme. PMID:21605678

Genistein and tyrphostin AG556 decrease ultra-rapidly activating delayed rectifier K+ current of human atria by inhibiting EGF receptor tyrosine kinase.

PubMed

Xiao, Guo-Sheng; Zhang, Yan-Hui; Wu, Wei; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2017-03-01

The ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. Human atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. The PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur . © 2017 The British Pharmacological Society.

Chronic myeloid leukemia progenitor cells require autophagy when leaving hypoxia-induced quiescence

PubMed Central

Ianniciello, Angela; Dumas, Pierre-Yves; Drullion, Claire; Guitart, Amélie; Villacreces, Arnaud; Peytour, Yan; Chevaleyre, Jean; Brunet de la Grange, Philippe; Vigon, Isabelle; Desplat, Vanessa; Priault, Muriel; Sbarba, Persio Dello; Ivanovic, Zoran; Mahon, François-Xavier; Pasquet, Jean-Max

2017-01-01

Albeit tyrosine kinase inhibitors anti-Abl used in Chronic Myeloid Leukemia (CML) block the deregulated activity of the Bcr-Abl tyrosine kinase and induce remission in 90% of patients, they do not eradicate immature hematopoietic compartments of leukemic stem cells. To elucidate if autophagy is important for stem cell survival and/or proliferation, we used culture in low oxygen concentration (0.1% O2 for 7 days) followed back by non-restricted O2 supply (normoxic culture) to mimic stem cell proliferation and commitment. Knockdown of Atg7 expression, a key player in autophagy, in K562 cell line inhibited autophagy compared to control cells. Upon 7 days at 0.1% O2 both K562 and K562 shATG7 cells stopped to proliferate and a similar amount of viable cells remained. Back to non-restricted O2 supply K562 cells proliferate whereas K562 shATG7 cells exhibited strong apoptosis. Using immunomagnetic sorted normal and CML CD34+ cells, we inhibited the autophagic process by lentiviral infection expressing shATG7 or using a Vps34 inhibitor. Both, normal and CML CD34+ cells either competent or deficient for autophagy stopped to proliferate in hypoxia. Surprisingly, while normal CD34+ cells proliferate back to non restricted O2 supply, the CML CD34+ cells deficient for autophagy failed to proliferate. All together, these results suggest that autophagy is required for CML CD34+ commitment while it is dispensable for normal CD34 cells. PMID:29228587

The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL

PubMed Central

Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

2016-01-01

Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML. PMID:27329306

The chimeric ubiquitin ligase SH2-U-box inhibits the growth of imatinib-sensitive and resistant CML by targeting the native and T315I-mutant BCR-ABL.

PubMed

Ru, Yi; Wang, Qinhao; Liu, Xiping; Zhang, Mei; Zhong, Daixing; Ye, Mingxiang; Li, Yuanchun; Han, Hua; Yao, Libo; Li, Xia

2016-06-22

Chronic myeloid leukemia (CML) is characterized by constitutively active fusion protein tyrosine kinase BCR-ABL. Although the tyrosine kinase inhibitor (TKI) against BCR-ABL, imatinib, is the first-line therapy for CML, acquired resistance almost inevitably emerges. The underlying mechanism are point mutations within the BCR-ABL gene, among which T315I is notorious because it resists to almost all currently available inhibitors. Here we took use of a previously generated chimeric ubiquitin ligase, SH2-U-box, in which SH2 from the adaptor protein Grb2 acts as a binding domain for activated BCR-ABL, while U-box from CHIP functions as an E3 ubiquitin ligase domain, so as to target the ubiquitination and degradation of both native and T315I-mutant BCR-ABL. As such, SH2-U-box significantly inhibited proliferation and induced apoptosis in CML cells harboring either the wild-type or T315I-mutant BCR-ABL (K562 or K562R), with BCR-ABL-dependent signaling pathways being repressed. Moreover, SH2-U-box worked in concert with imatinib in K562 cells. Importantly, SH2-U-box-carrying lentivirus could markedly suppress the growth of K562-xenografts in nude mice or K562R-xenografts in SCID mice, as well as that of primary CML cells. Collectively, by degrading the native and T315I-mutant BCR-ABL, the chimeric ubiquitin ligase SH2-U-box may serve as a potential therapy for both imatinib-sensitive and resistant CML.

Ibervillea sonorae (Cucurbitaceae) induces the glucose uptake in human adipocytes by activating a PI3K-independent pathway.

PubMed

Zapata-Bustos, Rocio; Alonso-Castro, Angel Josabad; Gómez-Sánchez, Maricela; Salazar-Olivo, Luis A

2014-03-28

Ibervillea sonorae (S. Watson) Greene (Cucurbitaceae), a plant used for the empirical treatment of type 2 diabetes in México, exerts antidiabetic effects on animal models but its mechanism of action remains unknown. The aim of this study is to investigate the antidiabetic mechanism of an Ibervillea sonorae aqueous extract (ISE). Non-toxic ISE concentrations were assayed on the glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes, both in the absence or the presence of insulin signaling pathway inhibitors, and on murine and human adipogenesis. Chemical composition of ISE was examined by spectrophotometric and HPLC techniques. ISE stimulated the 2-NBDGlucose uptake by mature adipocytes in a concentration-dependent manner. ISE 50 µg/ml induced the 2-NBDG uptake in insulin-sensitive 3T3-F442A, 3T3-L1 and human adipocytes by 100%, 63% and 33%, compared to insulin control. Inhibitors for the insulin receptor, PI3K, AKT and GLUT4 blocked the 2-NBDG uptake in murine cells, but human adipocytes were insensitive to the PI3K inhibitor Wortmannin. ISE 50 µg/ml also stimulated the 2-NBDG uptake in insulin-resistant adipocytes by 117% (3T3-F442A), 83% (3T3-L1) and 48% (human). ISE induced 3T3-F442A adipogenesis but lacked proadipogenic effects on 3T3-L1 and human preadipocytes. Chemical analyses showed the presence of phenolics in ISE, mainly an appreciable concentration of gallic acid. Ibervillea sonorae exerts its antidiabetic properties by means of hydrosoluble compounds stimulating the glucose uptake in human preadipocytes by a PI3K-independent pathway and without proadipogenic effects. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

PubMed

Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

2013-12-16

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

Vitamins K1 and K2: The Emerging Group of Vitamins Required for Human Health.

PubMed

Schwalfenberg, Gerry Kurt

2017-01-01

To review the evidence for the use of vitamin K supplementation in clinical conditions such as osteoporosis, vascular calcification, arthritis, cancer, renal calculi, diabetes, and warfarin therapy. PubMed was searched for articles on vitamin K (K1 and K2) along with books and conference proceedings and health conditions listed above. Level I and II evidence supports the use of vitamins K1 and K2 in osteoporosis and Level II evidence supports vitamin K2 in prevention of coronary calcification and cardiovascular disease. Evidence is insufficient for use in diabetes, arthritis, renal calculi, and cancer. Vitamin K2 may be a useful adjunct for the treatment of osteoporosis, along with vitamin D and calcium, rivaling bisphosphonate therapy without toxicity. It may also significantly reduce morbidity and mortality in cardiovascular health by reducing vascular calcification. Vitamin K2 appears promising in the areas of diabetes, cancer, and osteoarthritis. Vitamin K use in warfarin therapy is safe and may improve INR control, although a dosage adjustment is required. Vitamin K supplementation may be useful for a number of chronic conditions that are afflicting North Americans as the population ages. Supplementation may be required for bone and cardiovascular health.

Plasma stable, pH-sensitive non-ionic surfactant vesicles simultaneously enhance antiproliferative effect and selectivity of Sirolimus.

PubMed

Ghanbarzadeh, Saeed; Khorrami, Arash; Pourmoazzen, Zhaleh; Arami, Sanam

2015-05-01

The purpose of the present investigation was to prepare a plasma stable, pH-sensitive niosomal formulation to enhance Sirolimus efficacy and selectivity. pH-sensitive niosomal formulations bearing PEG-Poly (monomethyl itaconate)-CholC6 (PEG-PMMI-CholC6) copolymers and cholesteryl hemisuccinate (CHEMS) were prepared by a modified ethanol injection method and characterized with regard to pH-responsiveness and stability in human serum. The ability of pH-sensitive niosomes to enhance the Sirolimus cytotoxicity was evaluated in vitro using human erythromyeloblastoid leukemia cell line (K562) and compared with cytotoxicity effect on human umbilical vein endothelial cells (HUVEC). This study showed that both formulations can be rendered pH-sensitive property and were found to rapidly release their contents under mildly acidic conditions. However, the CHEMS-based niosomes lost their pH-sensitivity after incubation in plasma, whereas, PEG-PMMI-CholC6 niosomes preserved their ability to respond to pH change. Sirolimus encapsulated in pH-sensitive niosomes exhibited a higher cytotoxicity than the control conventional formulation on K562 cell line. On the other hand, both pH-sensitive niosomes showed lower antiproliferative effect on HUVEC cells. Plasma stable, pH-sensitive PEG-PMMI-CholC6-based niosomes can improve the in vitro efficiency and also reduce the side effects of Sirolimus.

Vitamins K1 and K2: The Emerging Group of Vitamins Required for Human Health

PubMed Central

2017-01-01

Objective To review the evidence for the use of vitamin K supplementation in clinical conditions such as osteoporosis, vascular calcification, arthritis, cancer, renal calculi, diabetes, and warfarin therapy. Quality of Evidence PubMed was searched for articles on vitamin K (K1 and K2) along with books and conference proceedings and health conditions listed above. Level I and II evidence supports the use of vitamins K1 and K2 in osteoporosis and Level II evidence supports vitamin K2 in prevention of coronary calcification and cardiovascular disease. Evidence is insufficient for use in diabetes, arthritis, renal calculi, and cancer. Main Message Vitamin K2 may be a useful adjunct for the treatment of osteoporosis, along with vitamin D and calcium, rivaling bisphosphonate therapy without toxicity. It may also significantly reduce morbidity and mortality in cardiovascular health by reducing vascular calcification. Vitamin K2 appears promising in the areas of diabetes, cancer, and osteoarthritis. Vitamin K use in warfarin therapy is safe and may improve INR control, although a dosage adjustment is required. Conclusion Vitamin K supplementation may be useful for a number of chronic conditions that are afflicting North Americans as the population ages. Supplementation may be required for bone and cardiovascular health. PMID:28698808

The Kell protein of the common K2 phenotype is a catalytically active metalloprotease, whereas the rare Kell K1 antigen is inactive. Identification of novel substrates for the Kell protein.

PubMed

Clapéron, Audrey; Rose, Christiane; Gane, Pierre; Collec, Emmanuel; Bertrand, Olivier; Ouimet, Tanja

2005-06-03

The Kell blood group is a highly polymorphic system containing over 20 different antigens borne by the protein Kell, a 93-kDa type II glycoprotein that displays high sequence homology with members of the M13 family of zinc-dependent metalloproteases whose prototypical member is neprilysin. Kell K1 is an antigen expressed in 9% of the Caucasian population, characterized by a point mutation (T193M) of the Kell K2 antigen, and located within a putative N-glycosylation consensus sequence. Recently, a recombinant, non-physiological, soluble form of Kell was shown to cleave Big ET-3 to produce the mature vasoconstrictive peptide. To better characterize the enzymatic activity of the Kell protein and the possible differences introduced by antigenic point mutations affecting post-translational processing, the membrane-bound forms of the Kell K1 and Kell K2 antigens were expressed either in K562 cells, an erythroid cell line, or in HEK293 cells, a non-erythroid system, and their pharmacological profiles and enzymatic specificities toward synthetic and natural peptides were evaluated. Results presented herein reveal that the two antigens possess considerable differences in their enzymatic activities, although not in their trafficking pattern. Indeed, although both antigens are expressed at the cell surface, Kell K1 protein is shown to be inactive, whereas the Kell K2 antigen binds neprilysin inhibitory compounds such as phosphoramidon and thiorphan with high affinity, cleaves the precursors of the endothelin peptides, and inactivates members of the tachykinin family with enzymatic properties resembling those of other members of the M13 family of metalloproteases to which it belongs.

Levcromakalim- and isoprenaline-induced relaxation of human isolated airways--role of the epithelium and of K+ channel activation.

PubMed

Black, J L; Johnson, P R; McKay, K O; Carey, D; Armour, C L

1994-06-01

In this study we have investigated the mechanism of action of levcromakalim and isoprenaline in human isolated airways with respect to the K+ channels they activate and the possibility that these smooth muscle relaxants activate K+ channels on the airway epithelium. Mechanical removal of the epithelial layer (mean percentage of epithelium present 20 +/- 3%, n = 20 tissues) did not affect the relaxation responses to levcromakalim or isoprenaline, either in terms of maximal relaxation or sensitivity. Whilst having no effect on isoprenaline-induced relaxation, studied from basal tone, the ATP-sensitive K+ channel blocker BRL 31660 (10, 30 and 50 microM) reduced relaxation responses induced (from basal tone) by levcromakalim from 74 +/- 6% (of the maximal response to isoprenaline) to 48 +/- 12% (n = 7), 9 +/- 9% (n = 4) and 0 (n = 4), respectively. Charybdotoxin, a blocker of high conductance Ca(2+)-activated K+ channels, at concentrations of 30 and 100 nM, had no effect on either levcromakalim- or or isoprenaline-induced relaxation responses and yet charybdotoxin was active at KCa channels in outside-out patches of hippocampal granule cells. Moreover, tetraethylammonium (10 mM) inhibited neither isoprenaline- nor levcromakalim-induced relaxation. This study has demonstrated that the relaxation responses elicited in human bronchus to isoprenaline and levcromakalim are likely to be the result of direct effects on the smooth muscle with no contribution from epithelial receptors or K+ channels. The actions of levcromakalim appear to be mediated only via activation of KATP channels. Further, we have made the important observation that, under the experimental conditions of our study, isoprenaline does not activate the KCa channel to produce relaxation in human bronchus.

Advancement of a 30K W Solar Electric Propulsion System Capability for NASA Human and Robotic Exploration Missions

NASA Technical Reports Server (NTRS)

Smith, Bryan K.; Nazario, Margaret L.; Manzella, David H.

2012-01-01

Solar Electric Propulsion has evolved into a demonstrated operational capability performing station keeping for geosynchronous satellites, enabling challenging deep-space science missions, and assisting in the transfer of satellites from an elliptical orbit Geostationary Transfer Orbit (GTO) to a Geostationary Earth Orbit (GEO). Advancing higher power SEP systems will enable numerous future applications for human, robotic, and commercial missions. These missions are enabled by either the increased performance of the SEP system or by the cost reductions when compared to conventional chemical propulsion systems. Higher power SEP systems that provide very high payload for robotic missions also trade favorably for the advancement of human exploration beyond low Earth orbit. Demonstrated reliable systems are required for human space flight and due to their successful present day widespread use and inherent high reliability, SEP systems have progressively become a viable entrant into these future human exploration architectures. NASA studies have identified a 30 kW-class SEP capability as the next appropriate evolutionary step, applicable to wide range of both human and robotic missions. This paper describes the planning options, mission applications, and technology investments for representative 30kW-class SEP mission concepts under consideration by NASA

Human endogenous retrovirus K Gag coassembles with HIV-1 Gag and reduces the release efficiency and infectivity of HIV-1.

PubMed

Monde, Kazuaki; Contreras-Galindo, Rafael; Kaplan, Mark H; Markovitz, David M; Ono, Akira

2012-10-01

Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.

Human Missions to Mars Orbit, Phobos, and Mars Surface Using 100-kWe-Class Solar Electric Propulsion

NASA Technical Reports Server (NTRS)

Price, Humphrey W.; Woolley, Ryan C.; Strange, Nathan J.; Baker, John D.

2014-01-01

Solar electric propulsion (SEP) tugs in the 100-kWe range, may be utilized to preposition cargo in the Mars system to enable more affordable human missions to Phobos and to the surface of Mars. The SEP tug, a high heritage follow-on to the 50-kWe SEP spacecraft proposed for the Asteroid Redirect Robotic Mission (ARRM), would have the same structure, tankage, electric propulsion components, and avionics as the ARRM version, But with double the number of solar arrays, Hall thrusters, and power processor units (PPUs) and would be accommodated within the same launch envelope defined for ARRM. As a feasibility study, a 950-day human mission to Phobos using a conjunction class trajectory, such as the 2033 opportunity, was developed using two 100-kWe SEP vehicles to preposition a habitat at Phobos and propulsion stages in high Mars orbit (HMO). An architecture concept for a crewed Mars surface lander mission was also developed as a reference to build on the Phobos mission architecture, adding a lander element that could be delivered using chemical propulsion and aerocapture.

KSC-97pc562

NASA Image and Video Library

1997-04-04

STS-83 Payload Specialist Roger K. Crouch is assisted into his launch/entry suit in the Operations and Checkout (O&C) Building. He is the Chief Scientist of the NASA Microgravity Space and Applications Division. He also has served as a Program Scientist for previous Spacelab microgravity missions and is an expert in semiconductor crystal growth. Since Crouch has more than 25 years of experience as a materials scientist, he will be concentrating on the five physics of materials processing experiments in the Middeck Glovebox Facility on the Blue shift. He will also share the workload with Thomas by monitoring the materials furnace experiments during this time. Crouch and six fellow crew members will shortly depart the O&C and head for Launch Pad 39A, where the Space Shuttle Columbia will lift off during a launch window that opens at 2:00 p.m. EST, April 4

Effect of Spaceflight on the Functions of NK and LAK Cells

NASA Technical Reports Server (NTRS)

Pierson, Duane L.; Grimm, Elizabeth A.; Pierson, Duane L.; Paloski, W. H. (Technical Monitor)

1999-01-01

Spaceflight-associated stress alters some aspects of the human immune response. In this study, we determined the effects of 10 days aboard the Space Shuttle on the cytotoxic activity of NK and LAK cells. The subjects of this study were crewmembers of two 10-day shuttle flights. Ten-ml blood specimens were obtained from ten astronauts 10 days before launch, immediately after landing, and 3 days after landing. PBMCs were separated from the blood specimens and stored at -800 C. All PBMCs were thawed simultaneously, and the cytotoxic activities of NK and LAK cells were measured by a 4-hour Cr-51 release assay. K562 cells were used to assess NK-cell cytotoxicity. After 4 days of IL-2 activation, the LAK cell cytotoxic activity was determined using K562 and Daudi cells as the target cells. NK-cell cytotoxicity was decreased at landing (p less than 0.0005) in 9/10 astronauts, and in most cases recovered to preflight levels by 3 days after landing; NK-cell cytotoxicity was increased in one astronaut at landing. LAK cytotoxic activity against K562 cells was decreased at landing in 6/10 astronauts (p=0.018), and activity against Daudi cells was decreased in 7/10 astronauts (p=0.01). Phenotyping of PBMCs and LAK cells showed alterations in some surface markers and adhesion molecules (CD1 1 b, CD1 1 c, CD1 1 a, CD1 6, L-Selectin and CD3). Thus spaceflight leads to a decrease in the functions of NK and LAK cells in most astronauts.

Quantification of the functional expression of the Ca2+ -activated K+ channel KCa 3.1 on microglia from adult human neocortical tissue.

PubMed

Blomster, Linda V; Strøbaek, Dorte; Hougaard, Charlotte; Klein, Jessica; Pinborg, Lars H; Mikkelsen, Jens D; Christophersen, Palle

2016-12-01

The K Ca 3.1 channel (KCNN4) is an important modulator of microglia responses in rodents, but no information exists on functional expression on microglia from human adults. We isolated and cultured microglia (max 1% astrocytes, no neurons or oligodendrocytes) from neocortex surgically removed from epilepsy patients and employed electrophysiological whole-cell measurements and selective pharmacological tools to elucidate functional expression of K Ca 3.1. The channel expression was demonstrated as a significant increase in the voltage-independent current by NS309, a K Ca 3.1/K Ca 2 activator, followed by full inhibition upon co-application with NS6180, a highly selective K Ca 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed K Ca 3.1, and the difference in current between full activation and inhibition (ΔK Ca 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals at least 585 channels per cell. Serial K Ca 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither changed the K Ca 3.1 current nor the fraction of K Ca 3.1 expressing cells. In contrast, the anti-inflammatory cytokine IL-4 slightly increased the K Ca 3.1 current per cell, but as the membrane area also increased, there was no significant change in channel density. A large fraction of the microglia also expressed a voltage-dependent current sensitive to the K Ca 1.1 modulators NS1619 and Paxilline and an inward-rectifying current with the characteristics of a K ir channel. The high functional expression of K Ca 3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065-2078. © 2016 Wiley Periodicals, Inc.

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies.

PubMed

Kelly, R A; O'Hara, D S; Canessa, M L; Mitch, W E; Smith, T W

1985-09-25

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.

Gamma reactivation using the spongy effect of KLF1-binding site sequence: an approach in gene therapy for beta-thalassemia

PubMed Central

Heydari, Nasrin; Shariati, Laleh; Khanahmad, Hossein; Hejazi, Zahra; Shahbazi, Mansoureh; Salehi, Mansoor

2016-01-01

Objective(s): β-thalassemia is one of the most common genetic disorders in the world. As one of the promising treatment strategies, fetal hemoglobin (Hb F) can be induced. The present study was an attempt to reactivate the γ-globin gene by introducing a gene construct containing KLF1 binding sites to the K562 cell line. Materials and Methods: A plasmid containing a 192 bp sequence with two repeats of KLF1 binding sites on β-globin and BCL11A promoters was constructed and used to transfect the K562 cell line. Positive selection was performed under treatment with 150 μg/ml hygromycin B. The remaining cells were expanded and harvested on day 28, and genomic DNA was extracted. The PCR was carried out to verify insertion of DNA fragment to the genome of K562 cells. The cells were differentiated with 15 μg/ml cisplatin. Flowcytometry was performed to identify erythroid differentiation by detection of CD235a+ cells. Real-time RT-PCR was performed to evaluate γ-globin expression in the transfected cells. Results: A 1700 bp fragment was observed on agarose gel as expected and insertion of DNA fragment to the genome of K562 cells was verified. Totally, 84% of cells were differentiated. The transfected cells significantly increased γ-globin expression after differentiation compared to untransfected ones. Conclusion: The findings demonstrate that the spongy effect of KLF1-binding site on BCL11A and β-globin promoters can induce γ-globin expression in K562 cells. This novel strategy can be promising for the treatment of β-thalassemia and sickle cell disease. PMID:27872702

The Promotion of Erythropoiesis via the Regulation of Reactive Oxygen Species by Lactic Acid

PubMed Central

Luo, Shun-Tao; Zhang, Dong-Mei; Qin, Qing; Lu, Lian; Luo, Min; Guo, Fu-Chun; Shi, Hua-Shan; Jiang, Li; Shao, Bin; Li, Meng; Yang, Han-Shuo; Wei, Yu-Quan

2017-01-01

The simultaneous increases in blood lactic acid and erythrocytes after intense exercise could suggest a link between lactate and the erythropoiesis. However, the effects of lactic acid on erythropoiesis remain to be elucidated. Here, we utilized a mouse model to determine the role of lactic acid in this process in parallel with studies using leukaemic K562 cells. Treatment of K562 cells in vitro with lactic acid increased the mRNA and protein expression of haemoglobin genes and the frequency of GPA+ cells. Also, increases in haematocrit and CD71−/Ter119+ erythroid cells were observed in lactic acid-treated mice, which showed a physiological increase in blood lactate. Mouse bone marrow CD34+/CD117− cells showed an increase in erythroid burst-forming units after stimulation with lactic acid in vitro. Furthermore, lactic acid increased the intracellular reactive oxygen species (ROS) content in bone marrow and in K562 cells. Erythroid differentiation induced in Haematopoietic Stem Cells (HSCs) and K562 cells by lactic acid was abolished by reducing ROS levels with SOD or 2-mercaptoethanol, which suggests that ROS is a critical regulator of this process. These findings provide a better understanding of the role of lactic acid in cellular metabolism and physiological functions. PMID:28165036

Inhibition of hyaluronic acid formation sensitizes chronic myelogenous leukemia to treatment with doxorubicin.

PubMed

Uchakina, Olga N; Ban, Hao; Hostetler, Bryan J; McKallip, Robert J

2016-11-01

In the current study we examined the ability of 4-methylumbelliferone (4-MU), which can inhibit hyaluronic acid synthesis, to sensitize K562 chronic myelogenous leukemia (CML) cells to doxorubicin therapy. Exposure of K562 cells to doxorubicin led to increased hyaluronic acid synthase (HAS) gene expression and increased levels of cell surface hyaluronic acid. Furthermore, exposure of K562 cells to exogenous HA caused resistance to doxorubicin-induced cell death. The combination of low dose 4-MU and doxorubicin led to increased apoptosis when compared to higher doses of any agent alone. Additionally, treatment with 4-MU led to a significant reduction in doxorubicin-induced increase in HA cell surface expression. Mechanistically, 4-MU treatment led to an increase in p38 activation and PARP cleavage. The role of p38 in 4-MU/doxorubicin-treated K562 cells was confirmed when p38 inhibitors led to protection from 4-MU/doxorubicin-induced apoptosis. Together, results from this study suggest that treatment with 4-MU increases the sensitivity of CML to chemotherapeutics by decreasing their HA-mediated resistance to apoptosis. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mapping of four distinct BCR-related loci to chromosome region 22q11: order of BCR loci relative to chronic myelogenous leukemia and acute lymphoblastic leukemia breakpoints

DOE Office of Scientific and Technical Information (OSTI.GOV)

Croce, C.M.; Huebner, K.; Isobe, M.

1987-10-01

A probe derived from the 3' region of the BCR gene (breakpoint cluster region gene) detects four distinct loci in the human genome. One of the loci corresponds to the complete BCR gene, whereas the other contain a 3' segment of the gene. After HindIII cleavage of human DNA, these four loci are detected as 23-, 19-, 13-, and 9-kikobase-pair fragments, designated BCR4, BCR3, BCR2, and BCR1, respectively, with BCR1 deriving from the original complete BCR gene. All four BCR loci segregate 100% concordantly with human chromosome 22 in a rodent-human somatic cell hybrid panel and are located at chromosomemore » region 22q11.2 by chromosomal in situ hybridization. The BCR2 and BCR4 loci are amplified in leukemia cell line K562 cells, indicating that they fall within the amplification unit that includes immunoglobulin lambda light chain locus (IGL) and ABL locus on the K562 Philadelphia chromosome (Ph/sup 1/). Similarly, in mouse-human hybrids retaining a Ph/sup 1/ chromosome derived from an acute lymphoblastic leukemia-in the absence of the 9q/sup +/ and 22, only BCR2 and BCR4 loci are retained. Thus, the order of loci on chromosome 22 is centromere ..-->.. BCR2, BCR4, and IGL ..-->.. BCR1 ..-->.. BCR3 ..-->.. SIS, possibly eliminating BCR2 and BCR4 loci as candidate targets for juxtaposition to the ABL gene in the acute lymphoblastic leukemia Ph/sup 1/ chromosome.« less

Tumorigenesis of K-ras mutation in human endometrial carcinoma via upregulation of estrogen receptor.

PubMed

Tu, Zheng; Gui, Liming; Wang, Jianliu; Li, Xiaoping; Sun, Pengming; Wei, Lihui

2006-05-01

To investigate the tumorigenesis of mutant [12Asp]-K-ras in endometrial carcinoma and its relationship with ER. We constructed pcDI-[12Asp]K-ras4B by inserting full-length [12Asp]K-ras4B from human endometrial carcinoma Hec-1A cells, into pcDI vector. Cell proliferation of NIH3T3 after transfection with pcDI-[12Asp]K-ras4B was measured by MTT assay. The cell transformation was determined by colony formation and tumor nodule development. [12Asp]-K-ras4B-NIH3T3 cells were transfected with constitutively active pCMV-RafCAAX and dominant-negative pCMV-RafS621A. Cell growth was measured by MTT assay and [3H]thymidine incorporation. After transfected with pcDI-[12Asp]K-ras4B or pCMV-RafS621A, the cells were harvested for Western blot and reporter assay to determine the expression and transcriptional activity of ERalpha and ERbeta, respectively. [12Asp]-K-ras4B enhanced NIH3T3 cells proliferation after 48 h post-transfection (P < 0.05). More colonies were grown 10 days after incubating pcDI-[12Asp]-K-ras4B-NIH3T3 cells (13.48%) than pcDI-NIH3T3 (4.26%) or untreated NIH3T3 (2.33%). The pcDI-[12Asp]-K-ras4B-NIH3T3 cells injected to the nude mice Balb/C developed tumor nodules with poor-differentiated cells after 12 days. An increase of ERalpha and ERbeta was observed in pcDI-[12Asp]-K-ras4B-NIH3T3 cells. RafS621A downregulated ERalpha and ERbeta expression. Estrogen induced the ER transcriptional activity by 5-fold in pcDI-NIH3T3 cells, 13-fold in pcDI-[12Asp]K-ras4B-NIH3T3 and 19-fold in HEC-1A. RafS621A suppressed the ER transcriptional activity. K-ras mutation induces tumorigenesis in endometrium, and this malignant transformation involves Raf signaling pathway and ER.

Antitumor effects of vitamins K1, K2 and K3 on hepatocellular carcinoma in vitro and in vivo.

PubMed

Hitomi, Misuzu; Yokoyama, Fumi; Kita, Yuko; Nonomura, Takako; Masaki, Tsutomu; Yoshiji, Hitoshi; Inoue, Hideyuki; Kinekawa, Fumihiko; Kurokohchi, Kazutaka; Uchida, Naohito; Watanabe, Seishiro; Kuriyama, Shigeki

2005-03-01

A number of studies have shown that various K vitamins, specifically vitamins K2 and K3, possess antitumor activity on various types of rodent- and human-derived neoplastic cell lines. In the present study, we examined the antitumor effects of vitamins K1, K2 and K3 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Furthermore, we examined the mechanisms of antitumor actions of these vitamins in vitro and in vivo. Although vitamin K1 did not inhibit proliferation of PLC/PRF/5 cells at a 90-microM concentration (the highest tested), vitamins K2 and K3 suppressed proliferation of the cells at concentrations of 90 and 9 microM, respectively. By flow cytometric analysis, it was shown that not only vitamin K1, but also vitamin K2 did not induce apoptosis or cell cycle arrest on PLC/PRF/5 cells. In contrast, vitamin K3 induced G1 arrest, but not apoptosis on PLC/PRF/5 cells. Subsequent in vivo study using subcutaneous HCC-bearing athymic nude mice demonstrated that both vitamins K2 and K3 markedly suppressed the growth of HCC tumors to similar extent. Protein expression of cyclin D1 and cyclin-dependent kinase 4 (Cdk4), but not p16INK4a Cdk inhibitor in the tumor was significantly reduced by vitamin K2 or K3 treatment, indicating that vitamins K2 and K3 may induce G1 arrest of cell cycle on PLC/PRF/5 cells in vivo. Taken collectively, vitamins K2 and K3 were able to induce potent antitumor effects on HCC in vitro and in vivo, at least in part, by inducing G1 arrest of the cell cycle. The results indicate that vitamins K2 and K3 may be useful agents for the treatment of patients with HCC.

The T-Cell Oncogene Tal2 Is a Target of PU.1 and Upregulated during Osteoclastogenesis

PubMed Central

Courtial, Nadine; Mücke, Christian; Herkt, Stefanie; Kolodziej, Stephan; Hussong, Helge; Lausen, Jörn

2013-01-01

Transcription factors play a crucial role in regulating differentiation processes during human life and are important in disease. The basic helix-loop-helix transcription factors Tal1 and Lyl1 play a major role in the regulation of gene expression in the hematopoietic system and are involved in human leukemia. Tal2, which belongs to the same family of transcription factors as Tal1 and Lyl1, is also involved in human leukaemia. However, little is known regarding the expression and regulation of Tal2 in hematopoietic cells. Here we show that Tal2 is expressed in hematopoietic cells of the myeloid lineage. Interestingly, we found that usage of the Tal2 promoter is different in human and mouse cells. Two promoters, hP1 and hP2 drive Tal2 expression in human erythroleukemia K562 cells, however in mouse RAW cells only the mP1 promoter is used. Furthermore, we found that Tal2 expression is upregulated during oesteoclastogenesis. We show that Tal2 is a direct target gene of the myeloid transcription factor PU.1, which is a key transcription factor for osteoclast gene expression. Strikingly, PU.1 binding to the P1 promoter is conserved between mouse and human, but PU.1 binding to P2 was only detected in human K562 cells. Additionally, we provide evidence that Tal2 influences the expression of the osteoclastic differentiation gene TRACP. These findings provide novel insight into the expression control of Tal2 in hematopoietic cells and reveal a function of Tal2 as a regulator of gene expression during osteoclast differentiation. PMID:24086757

The mapping of the human 52-kD Ro/SSA autoantigen gene to human chromosome II, and its polymorphisms