Enhancing the efficiency of direct reprogramming of human mesenchymal stem cells into mature neuronal-like cells with the combination of small molecule modulators of chromatin modifying enzymes, SMAD signaling and cyclic adenosine monophosphate levels.

PubMed

Alexanian, Arshak R; Liu, Qing-song; Zhang, Zhiying

2013-08-01

Advances in cell reprogramming technologies to generate patient-specific cells of a desired type will revolutionize the field of regenerative medicine. While several cell reprogramming methods have been developed over the last decades, the majority of these technologies require the exposure of cell nuclei to reprogramming large molecules via transfection, transduction, cell fusion or nuclear transfer. This raises several technical, safety and ethical issues. Chemical genetics is an alternative approach for cell reprogramming that uses small, cell membrane penetrable substances to regulate multiple cellular processes including cell plasticity. Recently, using the combination of small molecules that are involved in the regulation chromatin structure and function and agents that favor neural differentiation we have been able to generate neural-like cells from human mesenchymal stem cells. In this study, to improve the efficiency of neuronal differentiation and maturation, two specific inhibitors of SMAD signaling (SMAD1/3 and SMAD3/5/8) that play an important role in neuronal differentiation of embryonic stem cells, were added to our previous neural induction recipe. Results demonstrated that human mesenchymal stem cells grown in this culture conditions exhibited higher expression of several mature neuronal genes, formed synapse-like structures and exerted electrophysiological properties of differentiating neural stem cells. Thus, an efficient method for production of mature neuronal-like cells from human adult bone marrow derived mesenchymal stem cells has been developed. We concluded that specific combinations of small molecules that target specific cell signaling pathways and chromatin modifying enzymes could be a promising approach for manipulation of adult stem cell plasticity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Human gastrin-releasing peptide gene is located on chromosome 18.

PubMed

Naylor, S L; Sakaguchi, A Y; Spindel, E; Chin, W W

1987-01-01

Gastrin-releasing peptide (GRP), a bombesin-like peptide, increases plasma levels of gastrin, pancreatic polypeptide, glucagon, gastric inhibitory peptide, and insulin. GRP is produced in large quantities by small-cell lung cancer and acts as a growth factor for these cells. To determine if chromosomal changes in small-cell lung cancer are related to the expression of GRP, we chromosomally mapped the gene using human-mouse somatic cell hybrids. Twenty hybrids, characterized for human chromosomes, were analyzed by Southern filter hybridization of DNA digested with EcoRI. Human DNA cut with EcoRI yields a major band of 6.8 kb and a minor band of 11.3 kb. The 6.8 kb band segregated concordantly with chromosome 18 and the marker peptidase A. The chromosome 3 abnormalities seen in small-cell lung cancer do not correlate with the chromosomal location of GRP, suggesting that the elevated expression of this gene may be due to mechanisms other than chromosomal rearrangement.

THE CELLULAR ORIGIN OF HUMAN IMMUNOGLOBULINS (γ2, γ1M, γ1A)

PubMed Central

Mellors, Robert C.; Korngold, Leonhard

1963-01-01

A study was made of the cellular origin of human immunoglobulins (γ2, γ1M, γ1A). The results indicated that two closely related families of cells form immunoglobulins in human lymphoid tissue: germinal (reticular) centers and plasma cells. Thus their cellular origin in addition to their known antigenic relations further justifies placing the immunoglobulins in one family of proteins. Immunoglobulins were also formed to a small extent in primitive reticular cells which resembled those of germinal centers but were separated from them. Possibly such cells were undergoing transition to the much more numerous plasma cells with which they were commonly associated. The mantles of small lymphocytes which surrounded germinal centers did not contain detectable quantities of immunoglobulins. While in general only one type of immunoglobulin was present in an individual cell or germinal center, γ2- and γ1M-globulin were identified on occasion in the same plasma cell and germinal center. A peculiarity of the fetal thymus gland was the presence of immunoglobulin, mainly γ1M, in a small number of cells of small and intermediate size and primitive reticular appearance and in Hassall's corpuscles. PMID:14077999

Advanced Ring-Shaped Microelectrode Assay Combined with Small Rectangular Electrode for Quasi-In vivo Measurement of Cell-to-Cell Conductance in Cardiomyocyte Network

NASA Astrophysics Data System (ADS)

Nomura, Fumimasa; Kaneko, Tomoyuki; Hamada, Tomoyo; Hattori, Akihiro; Yasuda, Kenji

2013-06-01

To predict the risk of fatal arrhythmia induced by cardiotoxicity in the highly complex human heart system, we have developed a novel quasi-in vivo electrophysiological measurement assay, which combines a ring-shaped human cardiomyocyte network and a set of two electrodes that form a large single ring-shaped electrode for the direct measurement of irregular cell-to-cell conductance occurrence in a cardiomyocyte network, and a small rectangular microelectrode for forced pacing of cardiomyocyte beating and for acquiring the field potential waveforms of cardiomyocytes. The advantages of this assay are as follows. The electrophysiological signals of cardiomyocytes in the ring-shaped network are superimposed directly on a single loop-shaped electrode, in which the information of asynchronous behavior of cell-to-cell conductance are included, without requiring a set of huge numbers of microelectrode arrays, a set of fast data conversion circuits, or a complex analysis in a computer. Another advantage is that the small rectangular electrode can control the position and timing of forced beating in a ring-shaped human induced pluripotent stem cell (hiPS)-derived cardiomyocyte network and can also acquire the field potentials of cardiomyocytes. First, we constructed the human iPS-derived cardiomyocyte ring-shaped network on the set of two electrodes, and acquired the field potential signals of particular cardiomyocytes in the ring-shaped cardiomyocyte network during simultaneous acquisition of the superimposed signals of whole-cardiomyocyte networks representing cell-to-cell conduction. Using the small rectangular electrode, we have also evaluated the response of the cell network to electrical stimulation. The mean and SD of the minimum stimulation voltage required for pacing (VMin) at the small rectangular electrode was 166+/-74 mV, which is the same as the magnitude of amplitude for the pacing using the ring-shaped electrode (179+/-33 mV). The results showed that the addition of a small rectangular electrode into the ring-shaped electrode was effective for the simultaneous measurement of whole-cell-network signals and single-cell/small-cluster signals on a local site in the cell network, and for the pacing by electrical stimulation of cardiomyocyte networks.

Multifunctional gold nanocomposites designed for targeted CT/MR/optical trimodal imaging of human non-small cell lung cancer cells

NASA Astrophysics Data System (ADS)

Chen, Jingwen; Sun, Yingqi; Chen, Qian; Wang, Le; Wang, Suhe; Tang, Yun; Shi, Xiangyang; Wang, Han

2016-07-01

Multifunctional gold nanocomposites, which were designed as dendrimer-entrapped gold nanoparticles functionalized with gadolinium, cyanine dye (Cy5.5), and folic acid, were synthesized to be used as the first dendrimer-based clinical nanoprobes for targeted X-ray computed tomography/magnetic resonance/optical trimodal imaging in vitro and in vivo of human non-small cell cancer cells.Multifunctional gold nanocomposites, which were designed as dendrimer-entrapped gold nanoparticles functionalized with gadolinium, cyanine dye (Cy5.5), and folic acid, were synthesized to be used as the first dendrimer-based clinical nanoprobes for targeted X-ray computed tomography/magnetic resonance/optical trimodal imaging in vitro and in vivo of human non-small cell cancer cells. Electronic supplementary information (ESI) available: Synthesis and characterization data of the nanoprobes; biocompatibility results; confirmation of the tumor cell uptake of the nanoprobes in vitro and in vivo; biodistribution results in vivo. See DOI: 10.1039/c6nr03143a

Small Molecule Inhibition of cAMP Response Element Binding Protein in Human Acute Myeloid Leukemia Cells

PubMed Central

Mitton, Bryan; Chae, Hee-Don; Hsu, Katie; Dutta, Ritika; Aldana-Masangkay, Grace; Ferrari, Roberto; Davis, Kara; Tiu, Bruce C.; Kaul, Arya; Lacayo, Norman; Dahl, Gary; Xie, Fuchun; Li, Bingbing X.; Breese, Marcus R.; Landaw, Elliot M.; Nolan, Garry; Pellegrini, Matteo; Romanov, Sergei; Xiao, Xiangshu; Sakamoto, Kathleen M.

2016-01-01

The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy. PMID:27211267

Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells.

PubMed

Lin, Hui-Ping; Kuo, Li-Kuo; Chuu, Chih-Pin

2012-01-01

Curcumin (diferuloylmethane) is a phenolic compound present in turmeric and is ingested daily in many parts of the world. Curcumin has been reported to cause inhibition on proliferation and induction of apoptosis in many human cancer cell lines, including non-small cell lung cancer cells (NSCLC). However, the clinical application of curcumin is restricted by its low bioavailability. In this report, it was observed that combined treatment of a low dosage of curcumin (5-10 µM) with a low concentration (0.1-2.5 µM) of small molecule inhibitors, including AG1478, AG1024, PD173074, LY294002 and caffeic acid phenethyl ester (CAPE) increased the growth inhibition in two human NSCLC cell lines: A549 and H1299 cells. The observation suggested that combined treatment of a low dosage of curcumin with inhibitors against epidermal growth factor receptor (EGFR), insulin-like growth factor 1 (IGF-1R), fibroblast growth factors receptor (FGFR), phosphatidylinositol 3-kinases (PI3K) or NF-κB signaling pathway may be a potential adjuvant therapy beneficial to NSCLC patients. Copyright © 2011 John Wiley & Sons, Ltd.

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

PubMed

Josephson, A; Widenfalk, J; Trifunovski, A; Widmer, H R; Olson, L; Spenger, C

2001-11-12

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord. Copyright 2001 Wiley-Liss, Inc.

Rho GTPases and Regulation of Cell Migration and Polarization in Human Corneal Epithelial Cells

PubMed Central

Hou, Aihua; Toh, Li Xian; Gan, Kah Hui; Lee, Khee Jin Ryan; Manser, Edward; Tong, Louis

2013-01-01

Purpose Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. Methods Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. Results Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. Conclusion Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells. PMID:24130842

New medium used in the differentiation of human pluripotent stem cells to retinal cells is comparable to fetal human eye tissue.

PubMed

Wang, Xiaobing; Xiong, Kai; Lin, Cong; Lv, Lei; Chen, Jing; Xu, Chongchong; Wang, Songtao; Gu, Dandan; Zheng, Hua; Yu, Hurong; Li, Yan; Xiao, Honglei; Zhou, Guomin

2015-06-01

Human pluripotent stem cells (hPSCs) have the potential to differentiate along the retinal lineage. However, most induction systems are dependent on multiple small molecular compounds such as Dkk-1, Lefty-A, and retinoic acid. In the present study, we efficiently differentiated hPSCs into retinal cells using a retinal differentiation medium (RDM) without the use of small molecular compounds. This novel differentiation system recapitulates retinal morphogenesis in humans, i.e. hPSCs gradually differentiate into optic vesicle-shaped spheres, followed by optic cup-shaped spheres and, lastly, retinal progenitor cells. Furthermore, at different stages, hPSC-derived retinal cells mirror the transcription factor expression profiles seen in their counterparts during human embryogenesis. Most importantly, hinge epithelium was found between the hPSC-derived neural retina (NR) and retinal pigment epithelium (RPE). These data suggest that our culture system provides a new method for generating hPSC-derived retinal cells that, for the first time, might be used in human transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

NASA Astrophysics Data System (ADS)

Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

2013-12-01

Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives. Electronic supplementary information (ESI) available: ESI containing 1H NMR spectra and additional fibroblast characterization data. See DOI: 10.1039/c3nr04794f

Small Molecules Affect Human Dental Pulp Stem Cell Properties Via Multiple Signaling Pathways

PubMed Central

Al-Habib, Mey; Yu, Zongdong

2013-01-01

One fundamental issue regarding stem cells for regenerative medicine is the maintenance of stem cell stemness. The purpose of the study was to test whether small molecules can enhance stem cell properties of mesenchymal stem cells (MSCs) derived from human dental pulp (hDPSCs), which have potential for multiple clinical applications. We identified the effects of small molecules (Pluripotin (SC1), 6-bromoindirubin-3-oxime and rapamycin) on the maintenance of hDPSC properties in vitro and the mechanisms involved in exerting the effects. Primary cultures of hDPSCs were exposed to optimal concentrations of these small molecules. Treated hDPSCs were analyzed for their proliferation, the expression levels of pluripotent and MSC markers, differentiation capacities, and intracellular signaling activations. We found that small molecule treatments decreased cell proliferation and increased the expression of STRO-1, NANOG, OCT4, and SOX2, while diminishing cell differentiation into odonto/osteogenic, adipogenic, and neurogenic lineages in vitro. These effects involved Ras-GAP-, ERK1/2-, and mTOR-signaling pathways, which may preserve the cell self-renewal capacity, while suppressing differentiation. We conclude that small molecules appear to enhance the immature state of hDPSCs in culture, which may be used as a strategy for adult stem cell maintenance and extend their capacity for regenerative applications. PMID:23573877

Activation of the protein-tyrosine kinase associated with the bombesin receptor complex in small cell lung carcinomas

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

Gaudino, G.; Cirillo, D.; Naldini, L.

1988-04-01

It has been hypothesized that bombesin-like peptides produced by small cell lung carcinomas may sustain deregulated proliferation through an autocrine mechanism. The authors have shown that the neuropeptide bombesin leads to the activation of a protein-tyrosine kinase that phosphorylates a 115-kDa protein (p115) associated with the bombesin receptor complex in mouse Swiss 3T3 fibroblasts. They now report that phosphotyrosine antibodies recognize a 115-kDa protein, phosphorylated on tyrosine, in four human small cell lung carcinoma cell lines producing bombesin but not in a nonproducer variant line. p115 from detergent-treated small cell lung carcinoma cells binds to bombesin-Sepharose and can be phosphorylatedmore » on tyrosine in the presence of radiolabeled ATP and Mn{sup 2+}. As for the p115 immunoprecipitated from mouse fibroblast, the small cell lung carcinoma p115 can be phosphorylated in an immunocomplex kinase assay. However, the latter does not require the presence of exogenous bombesin for activity. Binding data, obtained by using radiolabeled ligand, suggest receptor occupancy in the cell lines producing bombesin. These observations are consistent with the hypothesis that proliferation in some human small cell lung carcinoma lines is under autocrine control, regulated through activation of bombesin receptors.« less

Time-and Concentration-Dependent Cytotoxicity of Ricin in Human Lung Epithelial Cells

DTIC Science & Technology

2007-07-01

lectin, ricin communis agglutinin, which is not directly cytotoxic but does have an affinity for red blood cells and can lead to agglutination and...Time- and Concentration-Dependent Cytotoxicity of Ricin in Human Lung Epithelial Cells Sharmaine Ramasamy and David Proll Human...Disease Control (CDC) Select Agent List. Using human small airway epithelial cells , this is the first study to investigate the time- and dose-dependent

Reaction-based small-molecule fluorescent probes for dynamic detection of ROS and transient redox changes in living cells and small animals.

PubMed

Lü, Rui

2017-09-01

Dynamic detection of transient redox changes in living cells and animals has broad implications for human health and disease diagnosis, because intracellular redox homeostasis regulated by reactive oxygen species (ROS) plays important role in cell functions, normal physiological functions and some serious human diseases (e.g., cancer, Alzheimer's disease, diabetes, etc.) usually have close relationship with the intracellular redox status. Small-molecule ROS-responsive fluorescent probes can act as powerful tools for dynamic detection of ROS and redox changes in living cells and animals through fluorescence imaging techniques; and great advances have been achieved recently in the design and synthesis of small-molecule ROS-responsive fluorescent probes. This article highlights up-to-date achievements in designing and using the reaction-based small-molecule fluorescent probes (with high sensitivity and selectivity to ROS and redox cycles) in the dynamic detection of ROS and transient redox changes in living cells and animals through fluorescence imaging. Copyright © 2017. Published by Elsevier Ltd.

Small-Molecule-Directed Hepatocyte-Like Cell Differentiation of Human Pluripotent Stem Cells.

PubMed

Mathapati, Santosh; Siller, Richard; Impellizzeri, Agata A R; Lycke, Max; Vegheim, Karianne; Almaas, Runar; Sullivan, Gareth J

2016-08-17

Hepatocyte-like cells (HLCs) generated in vitro from human pluripotent stem cells (hPSCs) provide an invaluable resource for basic research, regenerative medicine, drug screening, toxicology, and modeling of liver disease and development. This unit describes a small-molecule-driven protocol for in vitro differentiation of hPSCs into HLCs without the use of growth factors. hPSCs are coaxed through a developmentally relevant route via the primitive streak to definitive endoderm (DE) using the small molecule CHIR99021 (a Wnt agonist), replacing the conventional growth factors Wnt3A and activin A. The small-molecule-derived DE is then differentiated to hepatoblast-like cells in the presence of dimethyl sulfoxide. The resulting hepatoblasts are then differentiated to HLCs with N-hexanoic-Tyr, Ile-6 aminohexanoic amide (Dihexa, a hepatocyte growth factor agonist) and dexamethasone. The protocol provides an efficient and reproducible procedure for differentiation of hPSCs into HLCs utilizing small molecules. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

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

Sun, Yan; Xiao, Dong; Zhang, Ruo-Shuang

2007-06-15

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver.more » More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.« less

Gene selection and cancer type classification of diffuse large-B-cell lymphoma using a bivariate mixture model for two-species data.

PubMed

Su, Yuhua; Nielsen, Dahlia; Zhu, Lei; Richards, Kristy; Suter, Steven; Breen, Matthew; Motsinger-Reif, Alison; Osborne, Jason

2013-01-05

: A bivariate mixture model utilizing information across two species was proposed to solve the fundamental problem of identifying differentially expressed genes in microarray experiments. The model utility was illustrated using a dog and human lymphoma data set prepared by a group of scientists in the College of Veterinary Medicine at North Carolina State University. A small number of genes were identified as being differentially expressed in both species and the human genes in this cluster serve as a good predictor for classifying diffuse large-B-cell lymphoma (DLBCL) patients into two subgroups, the germinal center B-cell-like diffuse large B-cell lymphoma and the activated B-cell-like diffuse large B-cell lymphoma. The number of human genes that were observed to be significantly differentially expressed (21) from the two-species analysis was very small compared to the number of human genes (190) identified with only one-species analysis (human data). The genes may be clinically relevant/important, as this small set achieved low misclassification rates of DLBCL subtypes. Additionally, the two subgroups defined by this cluster of human genes had significantly different survival functions, indicating that the stratification based on gene-expression profiling using the proposed mixture model provided improved insight into the clinical differences between the two cancer subtypes.

A potential oral anticancer drug candidate, Moringa oleifera leaf extract, induces the apoptosis of human hepatocellular carcinoma cells

PubMed Central

JUNG, IL LAE; LEE, JU HYE; KANG, SE CHAN

2015-01-01

It has previously been reported that cold water-extracts of Moringa oleifera leaf have anticancer activity against various human cancer cell lines, including non-small cell lung cancer. In the present study, the anticancer activity of M. oleifera leaf extracts was investigated in human hepatocellular carcinoma HepG2 cells. By the analysis of apoptotic signals, including the induction of caspase or poly(ADP-ribose) polymerase cleavage, and the Annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, it was demonstrated that M. oleifera leaf extracts induce the apoptosis of HepG2 cells. In the hollow fiber assay, oral administration of the leaf extracts significantly reduced (44–52%) the proliferation of the HepG2 cells and A549 non-small cell lung cancer cells. These results support the potential of soluble extracts of M. oleifera leaf as orally administered therapeutics for the treatment of human liver and lung cancers. PMID:26622717

Preparation of pancreatic β-cells from human iPS cells with small molecules

PubMed Central

2012-01-01

Human induced pluripotent stem (iPS) cells obtained from patients are expected to be a useful source for cell transplantation therapy, because many patients (including those with type 1 diabetes and severe type 2 diabetes) are on waiting lists for transplantation for a long time due to the shortage of donors. At present, many concerns related to clinical application of human iPS cells have been raised, but rapid development of methods for the establishment, culture, and standardization of iPS cells will lead autologous cell therapy to be realistic sooner or later. However, establishment of a method for preparing some of desired cell types is still challenging. Regarding pancreatic β-cells, there have been many reports about differentiation of these cells from human embryonic stem (ES)/iPS cells, but a protocol for clinical application has still not been established. Since there is clear proof that cell transplantation therapy is effective for diabetes based on the results of clinical islet transplantation, pancreatic β-cells prepared from human iPS cells are considered likely to be effective for reducing the burden on patients. In this article, the current status of procedures for preparing pancreatic β-cells from human ES/iPS cells, including effective use of small molecules, is summarized, and some of the problems that still need to be overcome are discussed. PMID:22722666

The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells

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

Xu, Y.; Mirmalek-Sani, S.-H.; Yang, X.

2006-06-10

RNA interference (RNAi) has been used in functional genomics and offers innovative approaches in the development of novel therapeutics. Human mesenchymal stem cells offer a unique cell source for tissue engineering/regeneration strategies. The current study examined the potential of small interfering RNAs (siRNA) against human peroxisome proliferator activated receptor gamma (PPAR{gamma}) to suppress adipocyte differentiation (adipogenesis) in human preadipocytes and fetal-femur-derived mesenchymal cells. Adipogenesis was investigated using cellular and biochemical analysis. Transient transfection with PPAR{gamma}-siRNA using a liposomal-based strategy resulted in a significant inhibition of adipogenesis in human preadipocytes and fetal-femur-derived mesenchymal cells, compared to controls (cell, liposomal and negativemore » siRNA). The inhibitory effect of PPAR{gamma}-siRNA was supported by testing human PPAR{gamma} mRNA and adipogenic associated genes using reverse transcription polymerase chain reaction (RT-PCR) to adiponectin receptor 1 and 2 as well as examination of fatty acid binding protein 3 (FABP{sub 3}) expression, an adipocyte-specific marker. The current studies indicate that PPAR{gamma}-siRNA is a useful tool to study adipogenesis in human cells, with potential applications both therapeutic and in the elucidation of mesenchymal cell differentiation in the modulation of cell differentiation in human mesenchymal cells.« less

Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

PubMed Central

Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T.; Aftab, Blake T.; Armour, Michael; Gajula, Rajendra; Gandhi, Nishant; Salih, Tarek; Herman, Joseph M.; Wong, John; Rudin, Charles M.; Tran, Phuoc T.; Hales, Russell K.

2012-01-01

Purpose Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. Methods and Materials We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of KrasG12D-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. Results In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radio-sensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Conclusions Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer. PMID:23182391

Hedgehog pathway inhibition radiosensitizes non-small cell lung cancers.

PubMed

Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T; Aftab, Blake T; Armour, Michael; Gajula, Rajendra; Gandhi, Nishant; Salih, Tarek; Herman, Joseph M; Wong, John; Rudin, Charles M; Tran, Phuoc T; Hales, Russell K

2013-05-01

Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntag and radiation. In a transgenic mouse model of Kras(G12D)-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Hedgehog Pathway Inhibition Radiosensitizes Non-Small Cell Lung Cancers

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

Zeng, Jing; Aziz, Khaled; Chettiar, Sivarajan T.

2013-05-01

Purpose: Despite improvements in chemoradiation, local control remains a major clinical problem in locally advanced non-small cell lung cancer. The Hedgehog pathway has been implicated in tumor recurrence by promoting survival of tumorigenic precursors and through effects on tumor-associated stroma. Whether Hedgehog inhibition can affect radiation efficacy in vivo has not been reported. Methods and Materials: We evaluated the effects of a targeted Hedgehog inhibitor (HhAntag) and radiation on clonogenic survival of human non-small cell lung cancer lines in vitro. Using an A549 cell line xenograft model, we examined tumor growth, proliferation, apoptosis, and gene expression changes after concomitant HhAntagmore » and radiation. In a transgenic mouse model of Kras{sup G12D}-induced and Twist1-induced lung adenocarcinoma, we assessed tumor response to radiation and HhAntag by serial micro-computed tomography (CT) scanning. Results: In 4 human lung cancer lines in vitro, HhAntag showed little or no effect on radiosensitivity. By contrast, in both the human tumor xenograft and murine inducible transgenic models, HhAntag enhanced radiation efficacy and delayed tumor growth. By use of the human xenograft model to differentiate tumor and stromal effects, mouse stromal cells, but not human tumor cells, showed significant and consistent downregulation of Hedgehog pathway gene expression. This was associated with increased tumor cell apoptosis. Conclusions: Targeted Hedgehog pathway inhibition can increase in vivo radiation efficacy in lung cancer preclinical models. This effect is associated with pathway suppression in tumor-associated stroma. These data support clinical testing of Hedgehog inhibitors as a component of multimodality therapy for locally advanced non-small cell lung cancer.« less

Separation of human bone marrow by counterflow centrifugation monitored by DNA-flowcytometry.

PubMed

de Witte, T; Plas, A; Koekman, E; Blankenborg, G; Salden, M; Wessels, J; Haanen, C

1984-10-01

Human bone marrow was fractionated by counterflow centrifugation into 16 fractions with increasing cell size. Three distinct subpopulations could be recognized: small lymphocytic cells, medium-sized nucleated erythroid cells and large myeloid elements. DNA-flowcytometry and 3H-thymidine uptake showed that within the erythroid and myeloid cell populations counterflow centrifugation separates each population according to the cell cycle phase. Hypotonic treatment of bone marrow for removal of the erythroid nucleated cells resulted in a complete abrogation of the proliferating erythroid cell population. Counterflow centrifugation also separates the small non-proliferating myeloid and erythroid committed stem cells from the larger proliferating stem cells. It appeared feasible to separate the small lymphocytic cells from the majority of BFU-E and CFU-GM, due to the larger size of the proliferating normoblasts and the committed progenitor cells. Elimination of the mature lymphocytes from the haematopoietic stem cells by counterflow centrifugation may offer an alternative approach to the prevention of graft versus host disease (GvHD).

Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells.

PubMed

Yamada, Tadaaki; Bando, Hideaki; Takeuchi, Shinji; Kita, Kenji; Li, Qi; Wang, Wei; Akinaga, Shiro; Nishioka, Yasuhiko; Sone, Saburo; Yano, Seiji

2011-12-01

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. © 2011 Japanese Cancer Association.

[Occupational lung cancer. A comparison between humans and experimental animals].

PubMed

Adachi, S; Takemoto, K

1987-09-01

Many epidemiological and experimental studies have suggested that the respiratory tract is one of the most sensitive organs to environmental carcinogens. Nevertheless there is little evidence to determine the relationship between a specific environmental carcinogen and a cell type of lung cancer, because the cell types of lung cancer and their relative frequencies are highly complex compared with those of other organs and tissues. In the present paper, occupational lung-cancer characteristics, which are the clearest in the relation between cause and effect in human lung cancers, were reviewed in comparison with the results of animal experiments concerned with occupational lung carcinogens. Through accumulation of histopathological examinations of the lung cancer cases, the following relationships between cause and cell type were conjectured: chromium and squamous cell carcinoma; asbestos and adenocarcinoma; nickel and squamous cell carcinoma; beryllium and small cell carcinoma; bis (chloromethyl) ether and small cell carcinoma; mustard gas and squamous cell or small cell carcinoma; vinyl chloride and large cell or adenocarcinoma; radionuclides and small cell carcinoma. The relation pertaining to arsenic, benzotrichloride and tar could not be conjectured because of insufficient cases and information in the histological diagnosis. On the other hand, the carcinogenicity of these substances in occupational exposure has been confirmed by animal experiments administered intratracheally or by inhalation studies under relatively higher concentration. As a result of recent refinements of inhalation study, all-day and life-span exposure to extremely low concentrations, such as microgram/m3 orders, of certain substances has been possible. The characteristics of lung tumors occurring in these animals are rather different from those of human. For example, in mouse, almost all of the malignant lung tumors developed by carcinogens are adenocarcinomas and it is rare to find the squamous cell carcinoma. Moreover, small cell carcinoma and large cell carcinoma have not known to occur in the lungs of rats and mice. Therefore, future research should focus elucidating the specific relationship between cause and cell type of human lung cancer by means of animal experiments on lung cancer that give attention to the specificities of each experimental animal and the origin of the resultant lung tumor.

Small Molecule Protection of Bone Marrow Hematopoietic Stem Cells

DTIC Science & Technology

2017-12-01

using isogenic (mutant/complemented) human cell line pairs from patients with Fanconi anemia (FA), a heritable human bone marrow failure (BMF) syndrome ...small molecules could be therapeutically useful in reducing the risk of BMF in diseases such as Fanconi anemia, and perhaps after radiation exposure...damage-repair, DNA damage response, Fanconi anemia and associated bone marrow failure syndromes and environmental and molecular toxicology will all be

Derivation and Expansion Using Only Small Molecules of Human Neural Progenitors for Neurodegenerative Disease Modeling

PubMed Central

Reinhardt, Peter; Glatza, Michael; Hemmer, Kathrin; Tsytsyura, Yaroslav; Thiel, Cora S.; Höing, Susanne; Moritz, Sören; Parga, Juan A.; Wagner, Lydia; Bruder, Jan M.; Wu, Guangming; Schmid, Benjamin; Röpke, Albrecht; Klingauf, Jürgen; Schwamborn, Jens C.; Gasser, Thomas; Schöler, Hans R.; Sterneckert, Jared

2013-01-01

Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development. PMID:23533608

A basal stem cell signature identifies aggressive prostate cancer phenotypes

PubMed Central

Smith, Bryan A.; Sokolov, Artem; Uzunangelov, Vladislav; Baertsch, Robert; Newton, Yulia; Graim, Kiley; Mathis, Colleen; Cheng, Donghui; Stuart, Joshua M.; Witte, Owen N.

2015-01-01

Evidence from numerous cancers suggests that increased aggressiveness is accompanied by up-regulation of signaling pathways and acquisition of properties common to stem cells. It is unclear if different subtypes of late-stage cancer vary in stemness properties and whether or not these subtypes are transcriptionally similar to normal tissue stem cells. We report a gene signature specific for human prostate basal cells that is differentially enriched in various phenotypes of late-stage metastatic prostate cancer. We FACS-purified and transcriptionally profiled basal and luminal epithelial populations from the benign and cancerous regions of primary human prostates. High-throughput RNA sequencing showed the basal population to be defined by genes associated with stem cell signaling programs and invasiveness. Application of a 91-gene basal signature to gene expression datasets from patients with organ-confined or hormone-refractory metastatic prostate cancer revealed that metastatic small cell neuroendocrine carcinoma was molecularly more stem-like than either metastatic adenocarcinoma or organ-confined adenocarcinoma. Bioinformatic analysis of the basal cell and two human small cell gene signatures identified a set of E2F target genes common between prostate small cell neuroendocrine carcinoma and primary prostate basal cells. Taken together, our data suggest that aggressive prostate cancer shares a conserved transcriptional program with normal adult prostate basal stem cells. PMID:26460041

STK33 kinase activity is nonessential in KRAS-dependent cancer cells.

PubMed

Babij, Carol; Zhang, Yihong; Kurzeja, Robert J; Munzli, Anke; Shehabeldin, Amro; Fernando, Manory; Quon, Kim; Kassner, Paul D; Ruefli-Brasse, Astrid A; Watson, Vivienne J; Fajardo, Flordeliza; Jackson, Angela; Zondlo, James; Sun, Yu; Ellison, Aaron R; Plewa, Cherylene A; San, Miguel Tisha; Robinson, John; McCarter, John; Schwandner, Ralf; Judd, Ted; Carnahan, Josette; Dussault, Isabelle

2011-09-01

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors. ©2011 AACR.

Mesenchymal stem cell and derived exosome as small RNA carrier and Immunomodulator to improve islet transplantation.

PubMed

Wen, Di; Peng, Yang; Liu, Di; Weizmann, Yossi; Mahato, Ram I

2016-09-28

Human bone marrow mesenchymal stem cells (hBMSCs) and their exosomes can suppress immune reaction and deliver small RNAs. Thus, they may improve islet transplantation by delivering small RNAs for promoting islet function and inhibiting immune rejection. Here, we proposed an hBMSC and its exosome-based therapy to overcome immune rejection and poor islet function, both of which hinder the success of islet transplantation. We found overexpressed siFas and anti-miR-375 in plasmid encoding shFas and anti-miR-375 transfected hBMSC-derived exosomes, which silenced Fas and miR-375 of human islets and improved their viability and function against inflammatory cytokines. This plasmid transfected hBMSCs downregulated Fas and miR-375 of human islets in a humanized NOD scid gamma (NSG) mouse model, whose immune reaction was inhibited by injecting hBMSC and peripheral blood mononuclear cell (PBMC) co-cultured exosomes. These exosomes suppressed immune reaction by inhibiting PBMC proliferation and enhancing regulatory T cell (Treg) function. Collectively, our studies elucidated the mechanisms of RNA delivery from hBMSCs to human islets and the immunosuppressive effect of hBMSC and peripheral blood mononuclear cell co-cultured exosomes for improving islet transplantation. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of guanyl nucleotides on CCKB receptor binding in brain tissue and continuous cell lines: a comparative study.

PubMed

Kaufmann, R; Schöneberg, T; Henklein, P; Meyer, R; Martin, H; Ott, T

1995-07-01

The effects of non-hydrolyzable guanyl nucleotide analogue GTP-gamma S on CCKB receptor binding in human and guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells, rat glioma C6 cells and human small cell lung cancer NCI-H69 cells were investigated by using [3H]CCK-8S saturation and competition binding studies. GTP-gamma S caused inhibition of specific [3H]CCK-8S binding in a concentration dependent manner with a plateau at 10-25 microM. 25 microM GTP-gamma S resulted in a small but significant increase in Kd and IC50 values with amount very similar in all CCKB receptor models tested. However, the maximal number of specific [3H]CCK-8S binding sites (Bmax) was unaffected. Results suggest that CCKB receptors are G-protein coupled in a similar way to human and guinea-pig cortex, Jurkat cells, GH3 cells, C6 cells and NCI-H69 cells.

Characterization of a microdissection library from human chromosome region 3p14

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

Bardenheuer, W.; Szymanski, S.; Lux, A.

1994-01-15

Structural alterations in human chromosome region 3p14-p23 resulting in the inactivation of one or more tumor suppressor genes are thought to play a pathogenic role in small cell lung cancer, renal cell carcinoma, and other human neoplasms. To identify putative tumor suppressor genes, 428 recombinant clones from a microdissection library specific for human chromosome region 3p14 were isolated and characterized. Ninety-six of these (22.5%) were human single-copy DNA sequences, 57 of which were unique sequence clones. Forty-four of these were mapped to the microdissected region using a cell hybrid mapping panel. Within this mapping panel, four probes detected two newmore » chromosome breakpoints that were previously indistinguishable from the translocation breakpoint t(3;8) in 3p14.2 in hereditary renal cell carcinoma. One probe maps to the homozygously deleted region of the small cell lung cancer cell line U2020. In addition, microdissection clones have been shown to be suitable for isolation of yeast artificial chromosomes. 52 refs., 3 figs., 2 tabs.« less

Knockdown of Immature Colon Carcinoma Transcript 1 Inhibits Proliferation and Promotes Apoptosis of Non–Small Cell Lung Cancer Cells

PubMed Central

He, Jiantao; Zhang, Shenghui; Yang, Qingbo; Wang, Bo; Liu, Zhiyu; Wu, Xintian

2016-01-01

Non–small cell lung cancer, as the most frequent type lung cancer, has lower survival rate of 5 years, despite improvements in surgery and chemotherapy. Previous studies showed immature colon carcinoma transcript 1 is closely related to tumorigenesis of human cancer cells. In the present study, we found immature colon carcinoma transcript 1 was overexpressed in lung cancer tissues using Oncomine database mining, and the biological effect of immature colon carcinoma transcript 1 was investigated in non–small cell lung cancer cell lines 95D and A549. Lentivirus-mediated RNA interference was used to knock down immature colon carcinoma transcript 1 expression in 95D and A549 cells in vitro, and the knockdown efficiency was determined using quantitative real-time polymerase chain reaction and Western blot assay. Knockdown of immature colon carcinoma transcript 1 significantly suppressed non–small cell lung cancer cell proliferation and colony formation ability confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay. Flow cytometry was applied to measure cell cycle arrest, and the result showed the cell cycle arrested in G2/M phase in 95D cells and arrested in G0/G1 phase in A549 cells. Furthermore, we measured the levels of cell cycle–associated proteins by Western blot analysis and found immature colon carcinoma transcript 1–mediated cell proliferation inhibition appeared due to downregulation of cell cycle activator cyclin D1 and upregulation of cell cycle inhibitor p21. In addition, immature colon carcinoma transcript 1 silencing significantly induced non–small cell lung cancer cell apoptosis by annexin V/7-amino-actinomycin D double-staining assay. All our data suggest that immature colon carcinoma transcript 1 may play an important role for non–small cell lung cancer cell proliferation and could be a potential molecular target for diagnosing and treating human non–small cell lung cancer. PMID:27413166

Evaluation of role of Notch3 signaling pathway in human lung cancer cells.

PubMed

Hassan, Wael Abdo; Yoshida, Ryoji; Kudoh, Shinji; Motooka, Yamato; Ito, Takaaki

2016-05-01

There is still a debate on the extent to which Notch3 signaling is involved in lung carcinogenesis and whether such function is dependent on cancer type or not. To evaluate Notch3 expression in different types of human lung cancer cells. Notch3 was detected in human lung cancer cell lines and in tissues. Then, small interfering RNA (siRNA) was used to down-regulate the expression of Notch3 in H69AR small cell lung carcinoma (SCLC) cells; two non-small cell lung carcinoma (NSCLC) cells; A549 adenocarcinoma (ADC); and H2170 squamous cell carcinoma (SCC). In addition, Notch3 intracellular domain (N3ICD) plasmid was transfected into H1688 human SCLC cells. We observed the effect of deregulating Notch3 signaling on the following cell properties: Notch-related proteins, cell morphology, adhesion, epithelial-mesenchymal transition (EMT), motility, proliferation and neuroendocrine (NE) features of SCLC. Notch3 is mainly expressed in NSCLC, and the expression of Notch1, Hes1 and Jagged1 is affected by Notch3. Notch3 has opposite functions in SCLC and NSCLC, being a tumor suppressor in the former and tumor promoting in the latter, in the context of cell adhesion, EMT and motility. Regarding cell proliferation, we found that inhibiting Notch3 in NSCLC decreases cell proliferation and induces apoptosis in NSCLC. Notch3 has no effect on cell proliferation or NE features of SCLC. Notch3 signaling in lung carcinoma is dependent on cell type. In SCLC, Notch3 behaves as a tumor suppressor pathway, while in NSCLC it acts as a tumor-promoting pathway.

A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium.

PubMed

Wang, Yuli; Gunasekara, Dulan B; Reed, Mark I; DiSalvo, Matthew; Bultman, Scott J; Sims, Christopher E; Magness, Scott T; Allbritton, Nancy L

2017-06-01

The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation of Small SSEA-4-Positive Putative Stem Cells from the Ovarian Surface Epithelium of Adult Human Ovaries by Two Different Methods

PubMed Central

Virant-Klun, Irma; Skutella, Thomas; Hren, Matjaz; Gruden, Kristina; Cvjeticanin, Branko; Vogler, Andrej; Sinkovec, Jasna

2013-01-01

The adult ovarian surface epithelium has already been proposed as a source of stem cells and germinal cells in the literature, therefore it has been termed the “germinal epithelium”. At present more studies have confirmed the presence of stem cells expressing markers of pluripotency in adult mammalian ovaries, including humans. The aim of this study was to isolate a population of stem cells, based on the expression of pluripotency-related stage-specific embryonic antigen-4 (SSEA-4) from adult human ovarian surface epithelium by two different methods: magnetic-activated cell sorting and fluorescence-activated cell sorting. Both methods made it possible to isolate a similar, relatively homogenous population of small, SSEA-4-positive cells with diameters of up to 4 μm from the suspension of cells retrieved by brushing of the ovarian cortex biopsies in reproductive-age and postmenopausal women and in women with premature ovarian failure. The immunocytochemistry and genetic analyses revealed that these small cells—putative stem cells—expressed some primordial germ cell and pluripotency-related markers and might be related to the in vitro development of oocyte-like cells expressing some oocyte-specific transcription factors in the presence of donated follicular fluid with substances important for oocyte growth and development. The stemness of these cells needs to be further researched. PMID:23509763

Topographical distribution and morphology of NADPH-diaphorase-stained neurons in the human claustrum

PubMed Central

Hinova-Palova, Dimka V.; Edelstein, Lawrence; Landzhov, Boycho; Minkov, Minko; Malinova, Lina; Hristov, Stanislav; Denaro, Frank J.; Alexandrov, Alexandar; Kiriakova, Teodora; Brainova, Ilina; Paloff, Adrian; Ovtscharoff, Wladimir

2014-01-01

We studied the topographical distribution and morphological characteristics of NADPH-diaphorase-positive neurons and fibers in the human claustrum. These neurons were seen to be heterogeneously distributed throughout the claustrum. Taking into account the size and shape of stained perikarya as well as dendritic and axonal characteristics, Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd)-positive neurons were categorized by diameter into three types: large, medium and small. Large neurons ranged from 25 to 35 μm in diameter and typically displayed elliptical or multipolar cell bodies. Medium neurons ranged from 20 to 25 μm in diameter and displayed multipolar, bipolar and irregular cell bodies. Small neurons ranged from 14 to 20 μm in diameter and most often displayed oval or elliptical cell bodies. Based on dendritic characteristics, these neurons were divided into spiny and aspiny subtypes. Our findings reveal two populations of NADPHd-positive neurons in the human claustrum—one comprised of large and medium cells consistent with a projection neuron phenotype, the other represented by small cells resembling the interneuron phenotype as defined by previous Golgi impregnation studies. PMID:24904317

Effect of HSP27 on Human Breast Tumor Cell Growth and Motility

DTIC Science & Technology

1999-08-01

the small stress protein, HSP27 , on growth and motility characteristics of normal and tumor-derived human mammary cell lines. We hypothesized that...cells overexpressing HSP27 would show increased motility, altered chemotactic properties, increased resistance to heat killing and to certain drugs...Donna has prepared and studied 19 clonal MDA23 1 breast tumor cell lines that overexpress human HSP27 , and determined that, while heat resistance is

Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.

PubMed

Maruotti, Julien; Sripathi, Srinivas R; Bharti, Kapil; Fuller, John; Wahlin, Karl J; Ranganathan, Vinod; Sluch, Valentin M; Berlinicke, Cynthia A; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z; Bhutto, Imran; Lutty, Gerard A; Zack, Donald J

2015-09-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

PubMed Central

Maruotti, Julien; Sripathi, Srinivas R.; Bharti, Kapil; Fuller, John; Wahlin, Karl J.; Ranganathan, Vinod; Sluch, Valentin M.; Berlinicke, Cynthia A.; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z.; Bhutto, Imran; Lutty, Gerard A.; Zack, Donald J.

2015-01-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. PMID:26269569

Single-chain antibody-delivered Livin siRNA inhibits human malignant melanoma growth in vitro and in vivo.

PubMed

Wang, Hao; Yang, Yifei; Wang, Wei; Guan, Bing; Xun, Meng; Zhang, Hai; Wang, Ziling; Zhao, Yong

2017-05-01

Although gene therapy has brought new insights into the treatment of malignant melanoma, targeting delivery of nucleic acid which targets critical oncogene/anti-oncogene in vivo is still a bottleneck in the therapeutic application. Our previous in vitro studies have found that the oncogene Livin could serve as a potential molecular target by small interfering RNA for gene therapy of malignant melanoma. However, how to transport Livin small interfering RNA into malignant melanoma cells specifically and efficiently in vivo needs further investigation. Cumulative evidence has suggested that single-chain antibody-mediated small interfering RNA targeted delivery is an effective way to silence specific genes in human cancer cells. Indeed, this study designed a protamine-single-chain antibody fusion protein, anti-MM scFv-tP, to deliver Livin small interfering RNA into LiBr cells. Further experiments confirmed the induction of cell apoptosis and suppression of cell proliferation by anti-MM scFv-tP in LiBr cells, along with efficient silence of Livin gene both in vitro and in vivo. Altogether, our findings provide a feasible approach to transport Livin small interfering RNA to malignant melanoma cells which would be a new therapeutic strategy for combating malignant melanoma.

Reactive Oxygen Species Produced by Prostate Cancer Cells Cause Castrate-Resistant Cell Growth by Inducing B-cell Lymphotoxin Release

DTIC Science & Technology

2013-06-01

data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this...Gaussia luciferase reconstitution, high throughput screen, small molecule inhibitors, human prostate carcinoma cells, pharmacokinetcs, prostate cancer...cells. The increase in ROS levels is probably due to an induction of a polyamine oxidation pathway and specific small molecule inhibitors of this

Sophoraflavanone G induces apoptosis of human cancer cells by targeting upstream signals of STATs.

PubMed

Kim, Byung-Hak; Won, Cheolhee; Lee, Yun-Han; Choi, Jung Sook; Noh, Kum Hee; Han, Songhee; Lee, Haeri; Lee, Chang Seok; Lee, Dong-Sup; Ye, Sang-Kyu; Kim, Myoung-Hwan

2013-10-01

Aberrantly activated signal transducer and activator of transcription (STAT) proteins are implicated with human cancers and represent essential roles for cancer cell survival and proliferation. Therefore, the development of small-molecule inhibitors of STAT signaling bearing pharmacological activity has therapeutic potential for the treatment of human cancers. In this study, we identified sophoraflavanone G as a novel small-molecule inhibitor of STAT signaling in human cancer cells. Sophoraflavanone G inhibited tyrosine phosphorylation of STAT proteins in Hodgkin's lymphoma and tyrosine phosphorylation of STAT3 in solid cancer cells by inhibiting phosphorylation of the Janus kinase (JAK) proteins, Src family tyrosine kinases, such as Lyn and Src, Akt, and ERK1/2. In addition, sophoraflavanone G inhibited STAT5 phosphorylation in murine-bone-marrow-derived pro-B cells transfected with translocated Ets Leukemia (TEL)-JAKs and cytokine-induced rat pre-T lymphoma cells, as well as STAT5b reporter activity in TEL-JAKs and STAT5b reporter systems. Sophoraflavanone G also inhibited nuclear factor-κB (NF-κB) signaling in multiple myeloma cells. Furthermore, sophoraflavanone G inhibited cancer cell proliferation and induced apoptosis by regulating the expression of apoptotic and anti-apoptotic proteins. Our data suggest that sophoraflavanone G is a novel small-molecule inhibitor of STAT signaling by targeting upstream signals of STATs that may have therapeutic potential for cancers caused by persistently activated STAT proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

6-Shogaol, an active constituent of dietary ginger, induces autophagy by inhibiting the AKT/mTOR pathway in human non-small cell lung cancer A549 cells.

PubMed

Hung, Jen-Yu; Hsu, Ya-Ling; Li, Chien-Te; Ko, Ying-Chin; Ni, Wen-Chiu; Huang, Ming-Shyan; Kuo, Po-Lin

2009-10-28

This study is the first study to investigate the anticancer effect of 6-shogaol in human non-small cell lung cancer A549 cells. 6-Shogaol inhibited cell proliferation by inducing autophagic cell death, but not, predominantly, apoptosis. Pretreatment of cells with 3-methyladenine (3-MA), an autophagy inhibitor, suppressed 6-shogaol mediated antiproliferation activity, suggesting that induction of autophagy by 6-shogaol is conducive to cell death. We also found that 6-shogaol inhibited survival signaling through the AKT/mTOR signaling pathway by blocking the activation of AKT and downstream targets, including the mammalian target of rapamycin (mTOR), forkhead transcription factors (FKHR) and glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of both of mTOR's downstream targets, p70 ribosomal protein S6 kinase (p70S6 kinase) and 4E-BP1, was also diminished. Overexpression of AKT by AKT cDNA transfection decreased 6-shogaol mediated autophagic cell death, supporting inhibition of AKT beneficial to autophagy. Moreover, reduction of AKT expression by siRNA potentiated 6-shogaol's effect, also supporting inhibition of AKT beneficial to autophagy. Taken together, these findings suggest that 6-shogaol may be a promising chemopreventive agent against human non-small cell lung cancer.

Inhibitor of PI3K/Akt Signaling Pathway Small Molecule Promotes Motor Neuron Differentiation of Human Endometrial Stem Cells Cultured on Electrospun Biocomposite Polycaprolactone/Collagen Scaffolds.

PubMed

Ebrahimi-Barough, Somayeh; Hoveizi, Elham; Yazdankhah, Meysam; Ai, Jafar; Khakbiz, Mehrdad; Faghihi, Faezeh; Tajerian, Roksana; Bayat, Neda

2017-05-01

Small molecules as useful chemical tools can affect cell differentiation and even change cell fate. It is demonstrated that LY294002, a small molecule inhibitor of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, can inhibit proliferation and promote neuronal differentiation of mesenchymal stem cells (MSCs). The purpose of this study was to investigate the differentiation effect of Ly294002 small molecule on the human endometrial stem cells (hEnSCs) into motor neuron-like cells on polycaprolactone (PCL)/collagen scaffolds. hEnSCs were cultured in a neurogenic inductive medium containing 1 μM LY294002 on the surface of PCL/collagen electrospun fibrous scaffolds. Cell attachment and viability of cells on scaffolds were characterized by scanning electron microscope (SEM) and 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. The expression of neuron-specific markers was assayed by real-time PCR and immunocytochemistry analysis after 15 days post induction. Results showed that attachment and differentiation of hEnSCs into motor neuron-like cells on the scaffolds with Ly294002 small molecule were higher than that of the cells on tissue culture plates as control group. In conclusion, PCL/collagen electrospun scaffolds with Ly294002 have potential for being used in neural tissue engineering because of its bioactive and three-dimensional structure which enhances viability and differentiation of hEnSCs into neurons through inhibition of the PI3K/Akt pathway. Thus, manipulation of this pathway by small molecules can enhance neural differentiation.

Enrichment and characterization of cancer stem cells from a human non-small cell lung cancer cell line.

PubMed

Zhao, Changhong; Setrerrahmane, Sarra; Xu, Hanmei

2015-10-01

Tumor cells from the same origin comprise different cell populations. Among them, cancer stem cells (CSCs) have higher tumorigenicity. It is necessary to enrich CSCs to determine an effective way to suppress and eliminate them. In the present study, using the non-adhesive culture system, tumor spheres were successfully generated from human A549 non-small cell lung cancer (NSCLC) cell line within 2 weeks. Compared to A549 adherent cells, sphere cells had a higher self-renewal ability and increased resistance to cytotoxic drugs. Sphere cells were more invasive and expressed stem cell markers including octamer‑binding transcription factor 4 (Oct4) and sex-determining region Y-box 2 (Sox2) at high levels. CD133, a disputed marker of lung CSCs, was also upregulated. Tumor sphere cells showed higher tumorigenic ability in vivo, indicating that more CSCs were enriched in the sphere cells. More blood vessels were formed in the tumor generated by sphere cells suggesting the interaction between CSCs and blood vessel. A reliable model of enriching CSCs from the human A549 NSCLC cell line was established that was simple and cost-effective compared to other methods.

Antitumor activity of EGFR-specific CAR T cells against non-small-cell lung cancer cells in vitro and in mice.

PubMed

Li, He; Huang, Yao; Jiang, Du-Qing; Cui, Lian-Zhen; He, Zhou; Wang, Chao; Zhang, Zhi-Wei; Zhu, Hai-Li; Ding, Yong-Mei; Li, Lin-Fang; Li, Qiang; Jin, Hua-Jun; Qian, Qi-Jun

2018-02-07

Effective control of non-small-cell lung cancer (NSCLC) remains clinically challenging, especially during advanced stages of the disease. This study developed an adoptive T-cell treatment through expression of a chimeric antigen receptor (CAR) to target human epidermal growth factor receptor (EGFR) in NSCLC. We optimized the non-viral piggyBac transposon system to engineer human T cells for the expression of EGFR-CAR, consisting of EGFR scFv, transmembrane domain, and intracellular 4-1BB-CD3ζ signaling domains. The modified CAR T cells exhibited expansion capability and anticancer efficacy in a time- and antigen-dependent manner in vitro as well as regression of EGFR-positive human lung cancer xenografts in vivo. EGFR-CAR T therapy is a promising strategy to improve the efficacy and potency of the adoptive immunotherapy in NSCLC. Moreover, EGFR-CAR T therapy could become a clinical application for NSCLC patients in the future.

Genetic and small molecule inhibition of arylamine N-acetyltransferase 1 reduces anchorage-independent growth in human breast cancer cell line MDA-MB-231.

PubMed

Stepp, Marcus W; Doll, Mark A; Carlisle, Samantha M; States, J Christopher; Hein, David W

2018-04-01

Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer. © 2018 Wiley Periodicals, Inc.

Human papillomavirus molecular biology.

PubMed

Harden, Mallory E; Munger, Karl

Human papillomaviruses are small DNA viruses with a tropism for squamous epithelia. A unique aspect of human papillomavirus molecular biology involves dependence on the differentiation status of the host epithelial cell to complete the viral lifecycle. A small group of these viruses are the etiologic agents of several types of human cancers, including oral and anogenital tract carcinomas. This review focuses on the basic molecular biology of human papillomaviruses. Copyright © 2016 Elsevier B.V. All rights reserved.

High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation

PubMed Central

Gence, Rémi; Bouchenot, Catherine; Lajoie-Mazenc, Isabelle

2018-01-01

ABSTRACT The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains. PMID:29192060

Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo.

PubMed

Havens, Aaron M; Sun, Hongli; Shiozawa, Yusuke; Jung, Younghun; Wang, Jingcheng; Mishra, Anjali; Jiang, Yajuan; O'Neill, David W; Krebsbach, Paul H; Rodgerson, Denis O; Taichman, Russell S

2014-04-01

The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ∼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.

Development of a human adaptive immune system in cord blood cell-transplanted mice.

PubMed

Traggiai, Elisabetta; Chicha, Laurie; Mazzucchelli, Luca; Bronz, Lucio; Piffaretti, Jean-Claude; Lanzavecchia, Antonio; Manz, Markus G

2004-04-02

Because ethical restrictions limit in vivo studies of the human hemato-lymphoid system, substitute human to small animal xenotransplantation models have been employed. Existing models, however, sustain only limited development and maintenance of human lymphoid cells and rarely produce immune responses. Here we show that intrahepatic injection of CD34+ human cord blood cells into conditioned newborn Rag2-/-gammac-/- mice leads to de novo development of B, T, and dendritic cells; formation of structured primary and secondary lymphoid organs; and production of functional immune responses. This provides a valuable model to study development and function of the human adaptive immune system in vivo.

Small functional groups for controlled differentiation of hydrogel-encapsulated human mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Benoit, Danielle S. W.; Schwartz, Michael P.; Durney, Andrew R.; Anseth, Kristi S.

2008-10-01

Cell-matrix interactions have critical roles in regeneration, development and disease. The work presented here demonstrates that encapsulated human mesenchymal stem cells (hMSCs) can be induced to differentiate down osteogenic and adipogenic pathways by controlling their three-dimensional environment using tethered small-molecule chemical functional groups. Hydrogels were formed using sufficiently low concentrations of tether molecules to maintain constant physical characteristics, encapsulation of hMSCs in three dimensions prevented changes in cell morphology, and hMSCs were shown to differentiate in normal growth media, indicating that the small-molecule functional groups induced differentiation. To our knowledge, this is the first example where synthetic matrices are shown to control induction of multiple hMSC lineages purely through interactions with small-molecule chemical functional groups tethered to the hydrogel material. Strategies using simple chemistry to control complex biological processes would be particularly powerful as they could make production of therapeutic materials simpler, cheaper and more easily controlled.

Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system.

PubMed

Metzger, Marco; Bareiss, Petra M; Danker, Timm; Wagner, Silvia; Hennenlotter, Joerg; Guenther, Elke; Obermayr, Florian; Stenzl, Arnulf; Koenigsrainer, Alfred; Skutella, Thomas; Just, Lothar

2009-12-01

Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract. Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies. The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment. It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

Antiproliferative effects of small fruit juices on several cancer cell lines.

PubMed

Yoshizawa, Y; Kawaii, S; Urashima, M; Fukase, T; Sato, T; Tanaka, R; Murofushi, N; Nishimura, H

2000-01-01

Juices prepared from small fruits, mainly growing in the northern part of Japan, were studied in an attempt to explore the feasibility of an assay that screens cytotoxic properties. Screening of 43 small fruit juices indicated that Actinidia polygama Maxim., Rosa rugosa Thunb., Vaccinium smallii A. Gray and Sorbus sambucifolia Roem, strongly inhibited the proliferation of all cancer cell lines examined and yet these juices were substantially less cytotoxic toward normal human cell lines.

Attenuation Measurements of Cell Pellets Using Through Transmission

NASA Astrophysics Data System (ADS)

Vadas, Justin; Greene, Claudia; Grygotis, Emma; Kuhn, Stephen; Mahlalela, Sanele; Newland, Tinisha; Ovutmen, Idil; Herd, Maria-Teresa

2011-10-01

A better understanding of differences in ultrasound tissue characteristics (such as speed of sound, attenuation, and backscatter coefficients) of benign compared to malignant cells could lead to improved cancer detection and diagnosis. A narrow band technique for measuring ultrasonic speed of sound and attenuation of small biological materials was developed and tested. Several mechanical improvements were made to the system to drastically improve alignment, allowing for accurate measurements of small cell pellets. Narrow band attenuation measurements were made first with tissue-mimicking phantoms and then with three different types of cell pellets: Chinese hamster ovary cells, healthy human prostate cells, and cancerous human prostate cells. Attenuation and speed of sound results for all three cell types, as well as the culture medium and tissue mimicking phantoms, are presented for a frequency range of 5 to 25 MHz.

Bcl-XL small interfering RNA suppresses the proliferation of 5-fluorouracil-resistant human colon cancer cells.

PubMed

Zhu, Hongbo; Guo, Wei; Zhang, Lidong; Davis, John J; Teraishi, Fuminori; Wu, Shuhong; Cao, Xiaobo; Daniel, Jonathan; Smythe, W Roy; Fang, Bingliang

2005-03-01

5-Fluorouracil (5-FU) is commonly used to treat human colon cancers but resistance to this compound is frequently observed in clinics. To characterize mechanisms of resistance to 5-FU and to develop new strategies for overcoming it, we established two cell lines that were resistant to 5-FU but not other chemotherapeutic agents from parental 5-FU-sensitive cell lines. Western blot analysis revealed that these resistant cells overexpressed the proteins Bcl-XL, Bcl-Xs, and Bik, and further data showed that the cells were resistant to 5-FU-induced DNA damage and cell cycle disorder. However, in parental cells, enforced expression of Bcl-XL protein provided only limited protection from 5-FU-induced apoptosis and overexpression of Bcl-XL protein did not affect 5-FU-induced DNA damage or cell cycle changes; these findings suggested that overexpression of Bcl-XL protein was not the major contributor to 5-FU resistance in any of our cells lines. Even so, knockdown of Bcl-XL protein expression by Bcl-XL-specific small interfering RNA could inhibit proliferation more effectively in 5-FU-resistant cells than in 5-FU-sensitive cells, and the combination of Bcl-XL-specific small interfering RNA and 5-FU had additive effect on the inhibition of 5-FU-resistant cells. These results suggest that down-regulation of Bcl-XL protein expression might provide a new treatment strategy for human 5-FU-resistant colon cancer therapy.

The histone demethylase PHF8 is an oncogenic protein in human non-small cell lung cancer

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

Shen, Yuzhou; Pan, Xufeng; Zhao, Heng, E-mail: hengzhao1966@sina.com

2014-08-15

Highlights: • PHF8 overexpresses in human NSCLC and predicts poor survival. • PHF8 regulates lung cancer cell growth and transformation. • PHF8 regulates apoptosis in human lung cancer cells. • PHF8 promotes miR-21 expression in human lung cancer. • MiR-21 is critically essential for PHF8 function in human lung cancer cells. - Abstract: PHF8 is a JmjC domain-containing protein and erases repressive histone marks including H4K20me1 and H3K9me1/2. It binds to H3K4me3, an active histone mark usually located at transcription start sites (TSSs), through its plant homeo-domain, and is thus recruited and enriched in gene promoters. PHF8 is involved inmore » the development of several types of cancer, including leukemia, prostate cancer, and esophageal squamous cell carcinoma. Herein we report that PHF8 is an oncogenic protein in human non-small cell lung cancer (NSCLC). PHF8 is up-regulated in human NSCLC tissues, and high PHF8 expression predicts poor survival. Our in vitro and in vivo evidence demonstrate that PHF8 regulates lung cancer cell proliferation and cellular transformation. We found that PHF8 knockdown induces DNA damage and apoptosis in lung cancer cells. PHF8 promotes miR-21 expression in human lung cancer, and miR-21 knockdown blocks the effects of PHF8 on proliferation and apoptosis of lung cancer cells. In summary, PHF8 promotes lung cancer cell growth and survival by regulating miR-21.« less

Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules.

PubMed

Tasnim, Farah; Phan, Derek; Toh, Yi-Chin; Yu, Hanry

2015-11-01

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy, disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small molecule-based approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3'-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers, urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen, Chlorpromazine, Diclofenac, Digoxin, Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore, SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development. Copyright © 2015 Elsevier Ltd. All rights reserved.

Do EBV Encoded Small RNAs Interfere with Tumor Suppressor APC in EBV Associated Breast Cancers

DTIC Science & Technology

2006-08-01

acute infectious mononucleosis but ultimately establishes persistent lifetime latent infection. In all latently infected cells EBVexpresses two small non...human initially causes acute infectious mononucleosis and later establishes persistent lifetime latent infection. In all latently EBV-infected cells, only

Combined small-molecule inhibition accelerates the derivation of functional, early-born, cortical neurons from human pluripotent stem cells

PubMed Central

Qi, Yuchen; Zhang, Xin-Jun; Renier, Nicolas; Wu, Zhuhao; Atkin, Talia; Sun, Ziyi; Ozair, M. Zeeshan; Tchieu, Jason; Zimmer, Bastian; Fattahi, Faranak; Ganat, Yosif; Azevedo, Ricardo; Zeltner, Nadja; Brivanlou, Ali H.; Karayiorgou, Maria; Gogos, Joseph; Tomishima, Mark; Tessier-Lavigne, Marc; Shi, Song-Hai; Studer, Lorenz

2017-01-01

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions for the rapid differentiation of hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of 6 pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 days of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders. PMID:28112759

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

PubMed

Denton, P W; Nochi, T; Lim, A; Krisko, J F; Martinez-Torres, F; Choudhary, S K; Wahl, A; Olesen, R; Zou, W; Di Santo, J P; Margolis, D M; Garcia, J V

2012-09-01

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus.

PubMed

Green, Charlotte J; Charlton, Catriona A; Wang, Lai-Mun; Silva, Michael; Morten, Karl J; Hodson, Leanne

2017-12-01

Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable. Therefore, the aim of this work was to develop an alternative method to isolate hepatocytes from non-encapsulated and small samples of human liver tissue. Healthy tissue from 44 human liver resections were graded for steatosis and tissue weights between 7.8 and 600 g were used for hepatocyte isolations. Tissue was diced and underwent a two-step digestion (EDTA and collagenase). Red cell lysis buffer was used to prevent red blood cell contamination and toxicity. Isolated hepatocyte viability was determined by trypan blue exclusion. Western blot and biochemical analyses were undertaken to ascertain cellular phenotype and function. Liver tissue that weighed ≥50 g yielded significantly higher (P < 0.01) cell viability than tissue <50 g. Viable cells secreted urea and displayed the phenotypic hepatocyte markers albumin and cytochrome P450. Presence of steatosis in liver tissue or intra-hepatocellular triglyceride content had no effect on cell viability. This methodology allows for the isolation of viable primary human hepatocytes from small amounts of "healthy" resected liver tissue which are not suitable for perfusion. This work provides the opportunity to increase the utilisation of resection surplus tissue, and may ultimately lead to an increased number of in vitro cellular studies being undertaken using the gold-standard model of human primary hepatocytes.

Two pore channels control Ebolavirus host cell entry and are drug targets for disease treatment

PubMed Central

Sakurai, Yasuteru; Kolokoltsov, Andrey A.; Chen, Cheng-Chang; Tidwell, Michael W.; Bauta, William E.; Klugbauer, Norbert; Grimm, Christian; Wahl-Schott, Christian; Biel, Martin; Davey, Robert A.

2015-01-01

Ebolavirus causes sporadic outbreaks of lethal hemorrhagic fever in humans with no currently approved therapy. Cells take up Ebolavirus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebolavirus entry into host cells requires the endosomal calcium channels called two pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs or small molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule we tested, inhibited infection of human macrophages, the primary target of Ebolavirus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebolavirus infection and may be effective targets for antiviral therapy. PMID:25722412

Overexpression of SAMD9 suppresses tumorigenesis and progression during non small cell lung cancer

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

Ma, Qing; Yu, Tao; Ren, Yao-Yao

2014-11-07

Highlights: • SAMD9 is down-regulated in human non-small cell lung cancer (NSCLC). • Knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro. • Overexpression of SAMD9 suppressed proliferation and invasion in A549 cells in vitro. • Depletion of SAMD9 increases tumor formation in vivo. - Abstract: The Sterile Alpha Motif Domain-containing 9 (SAMD9) gene has been recently emphasized during the discovery that it is expressed at a lower level in aggressive fibromatosis and some cases of breast and colon cancer, however, the underlying mechanisms are poorly understood. Here, we found that SAMD9 ismore » down-regulated in human non-small cell lung cancer (NSCLC). Furthermore, knockdown of SAMD9 expression is increased the invasion, migration and proliferation in H1299 cells in vitro and overexpression of SAMD9 suppressed proliferation and invasion in A549 cells. Finally, depletion of SAMD9 increases tumor formation in vivo. Our results may provide a strategy for blocking NSCLC tumorigenesis and progression.« less

Induction of sensory neurons from neuroepithelial stem cells by the ISX9 small molecule

PubMed Central

Ali, Rouknuddin Qasim; Blomberg, Evelina; Falk, Anna; Ährlund-Richter, Lars; Ulfendahl, Mats

2016-01-01

Hearing impairment most often involves loss of sensory hair cells and auditory neurons. As this loss is permanent in humans, a cell therapy approach has been suggested to replace damaged cells. It is thus of interest to generate lineage restricted progenitor cells appropriate for cell based therapies. Human long-term self-renewing neuroepithelial stem (lt-NES) cell lines exhibit in vitro a developmental potency to differentiate into CNS neural lineages, and importantly lack this potency in vivo, i.e do not form teratomas. Small-molecules-driven differentiation is today an established route obtain specific cell derivatives from stem cells. In this study, we have investigated the effects of three small molecules SB431542, ISX9 and Metformin to direct differentiation of lt-NES cells into sensory neurons. Exposure of lt-NES cells to Metformin or SB431542 did not induce any marked induction of markers for sensory neurons. However, a four days exposure to the ISX9 small molecule resulted in reduced expression of NeuroD1 mRNA as well as enhanced mRNA levels of GATA3, a marker and important player in auditory neuron specification and development. Subsequent culture in the presence of the neurotrophic factors BDNF and NT3 for another seven days yielded a further increase of mRNA expression for GATA3. This regimen resulted in a frequency of up to 25-30% of cells staining positive for Brn3a/Tuj1. We conclude that an approach with ISX9 small molecule induction of lt-NES cells into auditory like neurons may thus be an attractive route for obtaining safe cell replacement therapy of sensorineural hearing loss. PMID:27335699

Inhibition of TGF-β Signaling Promotes Human Pancreatic β-Cell Replication

PubMed Central

Dhawan, Sangeeta; Dirice, Ercument; Kulkarni, Rohit N.

2016-01-01

Diabetes is associated with loss of functional pancreatic β-cells, and restoration of β-cells is a major goal for regenerative therapies. Endogenous regeneration of β-cells via β-cell replication has the potential to restore cellular mass; however, pharmacological agents that promote regeneration or expansion of endogenous β-cells have been elusive. The regenerative capacity of β-cells declines rapidly with age, due to accumulation of p16INK4a, resulting in limited capacity for adult endocrine pancreas regeneration. Here, we show that transforming growth factor-β (TGF-β) signaling via Smad3 integrates with the trithorax complex to activate and maintain Ink4a expression to prevent β-cell replication. Importantly, inhibition of TGF-β signaling can result in repression of the Ink4a/Arf locus, resulting in increased β-cell replication in adult mice. Furthermore, small molecule inhibitors of the TGF-β pathway promote β-cell replication in human islets transplanted into NOD-scid IL-2Rgnull mice. These data reveal a novel role for TGF-β signaling in the regulation of the Ink4a/Arf locus and highlight the potential of using small molecule inhibitors of TGF-β signaling to promote human β-cell replication. PMID:26936960

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

PubMed Central

Moody, Terry W.; Tashakkori, Nicole; Mantey, Samuel A.; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T.

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB2R), neuromedin B receptor (BB1R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki = 1.4–10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists. PMID:28785244

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists.

PubMed

Moody, Terry W; Tashakkori, Nicole; Mantey, Samuel A; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB 2 R), neuromedin B receptor (BB 1 R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB 1 R, BB 2 R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB 1 R, BB 2 R, and BRS-3 with similar affinity ( K i = 1.4-10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca 2+ in human lung cancer cells transfected with BB 1 R, BB 2 R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.

Curcumin inhibits interferon-{alpha} induced NF-{kappa}B and COX-2 in human A549 non-small cell lung cancer cells

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

Lee, Jeeyun; Im, Young-Hyuck; Jung, Hae Hyun

2005-08-26

The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-{alpha} treatment. The IFN-{alpha}-treated A549 cells showed increase in protein expression levels of NF-{kappa}B and COX-2. IFN-{alpha} induced NF-{kappa}B binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-{alpha}-induced COX-2 expression in A549 cells. Within 10 min, IFN-{alpha} rapidly induced the binding activity of a {gamma}-{sup 32}P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-{alpha}-induced activations of NF-{kappa}B and COX-2 were inhibited by the addition of curcumin in A549more » cells.« less

Detection of human cytomegalovirus DNA replication in non-permissive Vero and 293 cells.

PubMed

Ellsmore, Victoria; Reid, G Gordon; Stow, Nigel D

2003-03-01

Human cytomegalovirus (HCMV) displays an exceptionally restricted host range in tissue culture with human fibroblasts being the principal fully permissive system. Nevertheless, immediate early (IE) proteins are expressed following infection of many non-permissive cell types of human, simian and murine origin, and viral origin-dependent DNA synthesis has been reconstituted by transfection of plasmids into Vero cells, a non-permissive line from African green monkey. We have examined the accumulation of HCMV strain AD169 DNA, and the replication of transfected HCMV origin-containing plasmids, in infected Vero and human embryonic kidney 293 cells, which were previously reported to express the major IE protein in a small proportion of infected cells but to be non-permissive for viral DNA synthesis. In Vero cells accumulation of origin-containing plasmid but not viral DNA occurred, whilst in 293 cells both DNAs accumulated. Immunofluorescence experiments indicated that following infection with 3 p.f.u. per cell, a small fraction of both cell types expressed the UL44 DNA replication protein. Neither cell line, however, supported the generation of infectious progeny virus. These results suggest that IE proteins expressed in Vero and 293 cells can induce the synthesis of early proteins capable of functioning in viral DNA replication, but there is a failure in later events on the pathway to infectious virus production. This provides further support for transfected Vero cells being a valid system in which to study HCMV DNA synthesis, and suggests that 293 cells may also prove useful in similar experiments.

KRAS-mutation status dependent effect of zoledronic acid in human non-small cell cancer preclinical models

PubMed Central

Kenessey, István; Kói, Krisztina; Horváth, Orsolya; Cserepes, Mihály; Molnár, Dávid; Izsák, Vera; Dobos, Judit; Hegedűs, Balázs

2016-01-01

Background In non-small cell lung cancer (NSCLC) KRAS-mutant status is a negative prognostic and predictive factor. Nitrogen-containing bisphosphonates inhibit prenylation of small G-proteins (e.g. Ras, Rac, Rho) and thus may affect proliferation and migration. In our preclinical work, we investigated the effect of an aminobisphosphonate compound (zoledronic acid) on mutant and wild type KRAS-expressing human NSCLC cell lines. Results We confirmed that zoledronic acid was unable to inhibit the prenylation of mutant K-Ras unlike in the case of wild type K-Ras. In case of in vitro proliferation, the KRAS-mutant human NSCLC cell lines showed resistance to zoledronic acid wild-type KRAS-cells proved to be sensitive. Combinatory application of zoledronic acid enhanced the cytostatic effect of cisplatin. Zoledronic acid did not induce significant apoptosis. In xenograft model, zoledronic acid significantly reduced the weight of wild type KRAS-EGFR-expressing xenograft tumor by decreasing the proliferative capacity. Futhermore, zoledronic acid induced VEGF expression and improved in vivo tumor vascularization. Materials and methods Membrane association of K-Ras was examined by Western-blot. In vitro cell viability, apoptotic cell death and migration were measured in NSCLC lines with different molecular background. The in vivo effect of zoledronic acid was investigated in a SCID mouse subcutaneous xenograft model. Conclusions The in vitro and in vivo inhibitory effect of zoledronic acid was based on the blockade of cell cycle in wild type KRAS-expressing human NSCLC cells. The zoledronic acid induced vascularization supported in vivo cytostatic effect. Our preclinical investigation suggests that patients with wild type KRAS-expressing NSCLC could potentially benefit from aminobisphosphonate therapy. PMID:27780929

Generation of Spinal Motor Neurons from Human Pluripotent Stem Cells.

PubMed

Santos, David P; Kiskinis, Evangelos

2017-01-01

Human embryonic stem cells (ESCs) are characterized by their unique ability to self-renew indefinitely, as well as to differentiate into any cell type of the human body. Induced pluripotent stem cells (iPSCs) share these salient characteristics with ESCs and can easily be generated from any given individual by reprogramming somatic cell types such as fibroblasts or blood cells. The spinal motor neuron (MN) is a specialized neuronal subtype that synapses with muscle to control movement. Here, we present a method to generate functional, postmitotic, spinal motor neurons through the directed differentiation of ESCs and iPSCs by the use of small molecules. These cells can be utilized to study the development and function of human motor neurons in healthy and disease states.

A Simple and Efficient Method of Slow Freezing for Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

PubMed

Imaizumi, Keitaro; Iha, Momoe; Nishishita, Naoki; Kawamata, Shin; Nishikawa, Shinichi; Akuta, Teruo

2016-01-01

Protocols available for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells are very inefficient and laborious compared to those for the cryopreservation of murine ES/iPS cells or other general cell lines. While the vitrification method may be adequate when working with small numbers of human ES/iPS cells, it requires special skills and is unsuitable when working with large cell numbers. Here, we describe a simple and efficient method for the cryopreservation of hES/hiPS cells that is based on a conventional slow freezing method that uses a combination of Pronase/EDTA for Stem™ and CP-5E™ [final concentrations: 6 % hydroxyethyl starch, 5 % DMSO, and 5 % ethylene glycol in saline]. CP-5E™ is highly effective for the cryopreservation of small cell clumps produced by hES/hiPS colony detachment in the presence of Pronase and EDTA (Pronase/EDTA for Stem™, a formulation containing multiple digestive enzymes from Streptomyces griseus). This novel method would be quite useful for large-scale hES/iPS cell banking for use in clinical applications.

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation.

PubMed

Montoya, Misty M; Ansel, K Mark

2017-04-13

CD4 + T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from naïve CD4 + T cells in vitro by activating them in the presence of the polarizing cytokines IL-1β, IL-6, IL-23, and TGFβ. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, RORγt, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Bcl-2-independent induction of apoptosis by neuropeptide receptor antagonist in human small cell lung carcinoma cells.

PubMed

Matsumoto, Y; Kawatani, M; Simizu, S; Tanaka, T; Takada, M; Imoto, M

2000-01-01

The broad-spectrum antagonist of neuropeptide receptor, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P, induced apoptosis selectively in human small cell lung carcinoma (SCLC) cells, which express gastrin-releasing peptide receptor, but not in other types of tumor cells as well as normal cells. The addition of gastrin-releasing peptide or bombesin and the inhibitor of caspase-3 suppressed [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis. Moreover, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P-induced apoptosis was not suppressed by Bcl-2 over-expression. Thus, blockage of gastrin-releasing peptide receptor-mediated signaling may provide a novel therapeutic option in SCLC which has become resistant to conventional chemotherapeutic agents.

Molecular cloning and expression analysis of the canine chemokine receptor CCR9.

PubMed

Maeda, Shingo; Ohno, Koichi; Tsukamoto, Atsushi; Nakashima, Ko; Fukushima, Kenjiro; Goto-Koshino, Yuko; Fujino, Yasuhito; Tsujimoto, Hajime

2012-01-15

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD. Copyright © 2011 Elsevier B.V. All rights reserved.

Transcription factor fos-related antigen-2 induces progressive peripheral vasculopathy in mice closely resembling human systemic sclerosis.

PubMed

Maurer, Britta; Busch, Nicole; Jüngel, Astrid; Pileckyte, Margarita; Gay, Renate E; Michel, Beat A; Schett, Georg; Gay, Steffen; Distler, Jörg; Distler, Oliver

2009-12-08

Microvascular damage is one of the first pathological changes in systemic sclerosis. In this study, we investigated the role of Fos-related antigen-2 (Fra-2), a transcription factor of the activator protein-1 family, in the peripheral vasculopathy of systemic sclerosis and examined the underlying mechanisms. Expression of Fra-2 protein was significantly increased in skin biopsies of systemic sclerosis patients compared with healthy controls, especially in endothelial and vascular smooth muscle cells. Fra-2 transgenic mice developed a severe loss of small blood vessels in the skin that was paralleled by progressive skin fibrosis at 12 weeks of age. The reduction in capillary density was preceded by a significant increase in apoptosis in endothelial cells at week 9 as detected by immunohistochemistry. Similarly, suppression of Fra-2 by small interfering RNA prevented human microvascular endothelial cells from staurosporine-induced apoptosis and improved both the number of tubes and the cumulative tube lengths in the tube formation assay. In addition, cell migration in the scratch assay and vascular endothelial growth factor-dependent chemotaxis in a modified Boyden chamber assay were increased after transfection of human microvascular endothelial cells with Fra-2 small interfering RNA, whereas proliferation was not affected. Fra-2 is present in human systemic sclerosis and may contribute to the development of microvasculopathy by inducing endothelial cell apoptosis and by reducing endothelial cell migration and chemotaxis. Fra-2 transgenic mice are a promising preclinical model to study the mechanisms and therapeutic approaches of the peripheral vasculopathy in systemic sclerosis.

A unique cell-surface protein phenotype distinguishes human small-cell from non-small-cell lung cancer

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

Baylin, S.B.; Gazdar, A.F.; Minna, J.D.

1982-08-01

Radioiodination (/sup 125/I) and two-dimensional polyacrylamide gel electrophoresis was used to determine that small-(oat) cell lung carcinoma (SCC)-a tumor with neuroedocrine features-possesses a surface protein pattern distinct from the other types of lung cancer cells (squamous, adeno-, and large-cell undifferentiated carcinoma). Twelve distinguishing proteins, 40 to 70 kilodaltons (kDal), characterized four separate lines of SCC; three of these, designated E (60 kDal; pI = 7.3), S (30 kDal; pI = 6.0), and U 57 kDal; pI = 5.6), may be unique SCC gene products and were identified only in (/sup 35/S)methionine labeling of SCC and not in non-SCC or humanmore » fibroblasts. Two lines of adeno-, one of squamous, and one of undifferentiated large-cell lung carcinoma exhibited similar surface protein patterns to one another. Nine distinguishing proteins (40 to 100 kDal) and at least five large proteins (>100 kDal) were unique to these lines. The surface protein phenotypes for SCC and non-SCC were distinct from those for human lymphoblastoid cells and fibroblasts. However, the neuroendocrine features of SCC were further substantiated because 6 of the 12 distinguishing SCC surface proteins, including E and U, were identified on human neuroblastoma cells. The proteins identified should (i) help define differentiation steps for normal and neoplastic bronchial epithelial cells, (ii) prove useful in better classifying lung cancers, and (iii) be instrumental in tracing formation of neuroendocrine cells.« less

Small molecule screening with laser cytometry can be used to identify pro-survival molecules in human embryonic stem cells.

PubMed

Sherman, Sean P; Pyle, April D

2013-01-01

Differentiated cells from human embryonic stem cells (hESCs) provide an unlimited source of cells for use in regenerative medicine. The recent derivation of human induced pluripotent cells (hiPSCs) provides a potential supply of pluripotent cells that avoid immune rejection and could provide patient-tailored therapy. In addition, the use of pluripotent cells for drug screening could enable routine toxicity testing and evaluation of underlying disease mechanisms. However, prior to establishment of patient specific cells for cell therapy it is important to understand the basic regulation of cell fate decisions in hESCs. One critical issue that hinders the use of these cells is the fact that hESCs survive poorly upon dissociation, which limits genetic manipulation because of poor cloning efficiency of individual hESCs, and hampers production of large-scale culture of hESCs. To address the problems associated with poor growth in culture and our lack of understanding of what regulates hESC signaling, we successfully developed a screening platform that allows for large scale screening for small molecules that regulate survival. In this work we developed the first large scale platform for hESC screening using laser scanning cytometry and were able to validate this platform by identifying the pro-survival molecule HA-1077. These small molecules provide targets for both improving our basic understanding of hESC survival as well as a tool to improve our ability to expand and genetically manipulate hESCs for use in regenerative applications.

5-(Furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one oxime (6f), a new synthetic compound, causes human fibrosarcoma HT-1080 cell apoptosis by disrupting tubulin polymerisation and inducing G2/M arrest.

PubMed

Zuo, Daiying; Pang, Lili; Shen, Jiwei; Guan, Qi; Bai, Zhaoshi; Zhang, Huijuan; Li, Yao; Lu, Guodong; Zhang, Weige; Wu, Yingliang

2017-06-01

In the current study, we synthesized a series of new compounds targeting tubulin and tested their anti-proliferative activities. Among these new synthetic com-pounds, 5-(furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one oxime (6f) exhibited significant anti-proliferative activity against different human cancer cell lines including human gastric adenocarcinoma SGC-7901, human non-small cell lung cancer A549, and human fibrosarcoma HT-1080. As a result, 6f was selected to further test the sensitivity to different cancer cell lines including human cervical cancer cell line HeLa, human breast cancer cell line MCF-7, non-small cell lung cancer cell line A549, human liver carcinoma cell line HepG-2, human oral squamous cell carcinoma cell lines KB, SGC-7901 and HT-1080. Among these cell lines, HT-1080 and HeLa are the most sensitive. Therefore, HT-1080 was selected to further explore the properties of anti-proliferative activity and the underlying mechanisms. Our data proved that 6f exhibited strong anti-proliferative effects against HT-1080 cells in a time- and dose-dependent manner. We showed that the growth inhibitory effect of 6f in HT-1080 cells was related with microtubule depolymerisation. Molecular docking studies revealed that 6f interacted and bound efficiently with the colchicine-binding site of tubulin. In addition, 6f treatment induced G2/M cell cycle arrest dose-dependently and subsequently induced cell apoptosis. Western blot study indicated that upregulation of cyclin B1 and p-cdc2 was related with G2/M arrest. 6f-induced cell apoptosis was associated with both mitochondrial and death receptor pathway. In conclusion, our data showed that 6f, among the newly synthetic compounds, exhibited highest anti-proliferative activity by disrupting the microtubule polymerisation, causing G2/M arrest and subsequently inducing cell apoptosis in HT-1080 cells. Hence, 6f is a promising microtubule depolymerising agent for the treatment of various cancers especially human fibrosarcoma.

How many TCR clonotypes does a body maintain?

PubMed Central

Lythe, Grant; Callard, Robin E.; Hoare, Rollo L.; Molina-París, Carmen

2016-01-01

We consider the lifetime of a T cell clonotype, the set of T cells with the same T cell receptor, from its thymic origin to its extinction in a multiclonal repertoire. Using published estimates of total cell numbers and thymic production rates, we calculate the mean number of cells per TCR clonotype, and the total number of clonotypes, in mice and humans. When there is little peripheral division, as in a mouse, the number of cells per clonotype is small and governed by the number of cells with identical TCR that exit the thymus. In humans, peripheral division is important and a clonotype may survive for decades, during which it expands to comprise many cells. We therefore devise and analyse a computational model of homeostasis of a multiclonal population. Each T cell in the model competes for self pMHC stimuli, cells of any one clonotype only recognising a small fraction of the many subsets of stimuli. A constant mean total number of cells is maintained by a balance between cell division and death, and a stable number of clonotypes by a balance between thymic production of new clonotypes and extinction of existing ones. The number of distinct clonotypes in a human body may be smaller than the total number of naive T cells by only one order of magnitude. PMID:26546971

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

PubMed

Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

2016-06-15

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. © 2016 UICC.

Macrophages and Dendritic Cells Are the Predominant Cells Infected in Measles in Humans

PubMed Central

Allen, Ingrid V.; McQuaid, Stephen; Penalva, Rosana; Ludlow, Martin; Duprex, W. Paul

2018-01-01

ABSTRACT Characterization of human measles cases is essential in order to better assess the data generated in model systems of morbillivirus infection. To this end, we collected formalin-fixed tissue samples from 23 natural measles cases from different areas in the world and different phases of disease ranging from prodromal and acute measles to a persistent infection in an immunocompromised subject. We show that the vast majority of measles virus (MV)-infected cells in epithelia were intraepithelial immune cells that were, in most cases, positive for the CD11c myeloid cell marker. Small numbers of measles virus-infected cytokeratin-positive epithelial cells were also detected in bronchial and appendix epithelia. Dissolution and disruption of uninfected and MV-infected alveolar and bronchial epithelia were prominent features of the measles cases, especially in the established and late phases of the disease. In some instances, this was associated with the formation of MV-infected multinucleated giant cells which expressed CD11c and/or macrophage cell marker 68, a pathological feature also prominently observed in closely associated mucosa-associated lymphoid tissue. Collectively, these data show that resident and inflammatory infiltrating immune cells alter the architecture of respiratory tract epithelia and highlight the necessity for additional research into the function(s) and expression of nectin-4 in human tissues. IMPORTANCE We have brought together a unique collection of 23 human cases of measles infection and studied the types of cells that are infected. This work has not been done with modern technologies such as double labeling with antibodies and confocal microscopy in human cases primarily due to the fact that it is difficult to obtain the material because, fortunately, measles is fatal in only a very small fraction of infected patients. During the past decades, the receptors for measles virus have been elucidated and monkey models have been developed. We found that, in most cases, independently of whether the tissues were obtained early or later in the infection, the primary cell types that were infected were those of the immune system such as lymphocytes, macrophages, and dendritic cells. A very small number of epithelial cells were also found to be infected. PMID:29743202

Hanging drop culture enhances differentiation of human adipose-derived stem cells into anterior neuroectodermal cells using small molecules.

PubMed

Amirpour, Noushin; Razavi, Shahnaz; Esfandiari, Ebrahim; Hashemibeni, Batoul; Kazemi, Mohammad; Salehi, Hossein

2017-06-01

Inspired by in vivo developmental process, several studies were conducted to design a protocol for differentiating of mesenchymal stem cells into neural cells in vitro. Human adipose-derived stem cells (hADSCs) as mesenchymal stem cells are a promising source for this purpose. At current study, we applied a defined neural induction medium by using small molecules for direct differentiation of hADSCs into anterior neuroectodermal cells. Anterior neuroectodermal differentiation of hADSCs was performed by hanging drop and monolayer protocols. At these methods, three small molecules were used to suppress the BMP, Nodal, and Wnt signaling pathways in order to obtain anterior neuroectodermal (eye field) cells from hADSCs. After two and three weeks of induction, the differentiated cells with neural morphology expressed anterior neuroectodermal markers such as OTX2, SIX3, β-TUB III and PAX6. The protein expression of such markers was confirmed by real time, RT-PCR and immunocytochemistry methods According to our data, it seems that the hanging drop method is a proper approach for neuroectodermal induction of hADSCs. Considering wide availability and immunosuppressive properties of hADSCs, these cells may open a way for autologous cell therapy of neurodegenerative disorders. Copyright © 2017 ISDN. Published by Elsevier Ltd. All rights reserved.

The Broad Institute: Screening for Dependencies in Cancer Cell Lines Using Small Molecules | Office of Cancer Genomics

Cancer.gov

Using cancer cell-line profiling, we established an ongoing resource to identify, as comprehensively as possible, the drug-targetable dependencies that specific genomic alterations impart on human cancers. We measured the sensitivity of hundreds of genetically characterized cancer cell lines to hundreds of small-molecule probes and drugs that have highly selective interactions with their targets, and that collectively modulate many distinct nodes in cancer cell circuitry.

Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway.

PubMed

Ramezanpour, Mahnaz; da Silva, Karen Burke; Sanderson, Barbara J S

2014-03-01

Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer.

Characterization of Nipah virus infection in a model of human airway epithelial cells cultured at an air-liquid interface.

PubMed

Escaffre, Olivier; Borisevich, Viktoriya; Vergara, Leoncio A; Wen, Julie W; Long, Dan; Rockx, Barry

2016-05-01

Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission.

Polarized Ends of Human Macula Densa Cells: Ultrastructural Investigation and Morphofunctional Correlations.

PubMed

Cangiotti, Angela Maria; Lorenzi, Teresa; Zingaretti, Maria Cristina; Fabri, Mara; Morroni, Manrico

2018-05-01

The morphology of the kidney macula densa (MD) has extensively been investigated in animals, whereas human studies are scanty. We studied the fine structure of human MD cells focusing on their apical and basal ends and correlating structure and function. The MD region was examined by transmission electron microscopy in six renal biopsies from patients with kidney disease. Ultrastructural analysis of MD cells was performed on serial sections. MD cells show two polarized ends. The apical portion is characterized by a single, immotile cilium associated with microvilli; apically, cells are joined by adhering junctions. In the basal portion, the cytoplasm contains small, dense granules and numerous, irregular cytoplasmic projections extending to the adjacent extraglomerular mesangium. The projections often contain small, dense granules. A reticulated basement membrane around MD cells separates them from the extraglomerular mesangium. Although the fact that tissue specimens came from patients with kidney disease mandates extreme caution, ultrastructural examination confirmed that MD cells have sensory features due to the presence of the primary cilium, that they are connected by apical adhering junctions forming a barrier that separates the tubular flow from the interstitium, and that they present numerous basal interdigitations surrounded by a reticulated basement membrane. Conceivably, the latter two features are related to the functional activity of the MD. The small, dense granules in the basal cytoplasm and in cytoplasmic projections are likely related to the paracrine function of MD cells. Anat Rec, 301:922-931, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

3-(4-chlorophenyl)-[1, 2, 3] oxadiazol-3-ium-5-olate and its 4-formyl analog-Ultrasound assisted synthesis and in-vitro anticancer evaluation against human tumor cell lines.

PubMed

Bhosale, Sachin K; Deshpande, Shreenivas R; Wagh, Rajendra D

2017-03-01

The title compound, 3-(4-chlorophenyl)-4-formyl-[1, 2, 3] oxadiazol-3-ium-5-olate 5 was synthesized under ultrasonication by formylation of 3-(4-chlorophenyl)-[1, 2, 3] oxadiazol-3-ium-5-olate 4 and characterized by spectral studies. The ultrasonic method of synthesis was found to be simple, ecofriendly, economical, reduces reaction time and gave good yield when compared with traditional methods of synthesis. Anticancer activity of the compounds were tested against 60 human tumor cell lines and compared with standard drug vincristine sulphate. Compound 5 was found to be active against CNS (SNB-75, %GI=46.71), renal (UO-31, %GI=31.52), non small cell lung (NCI-H522, %GI=25.65), leukemia (MOLT-4, %GI=23.02) human tumor cell lines whereas, compound 4 against breast (MDA-MB-231/ATCC, %GI=19.90, T-47D %GI=16.50, MCF-7 15.10) and ovarian (IGROV1 %GI=19.30, OVCAR-4 %GI=17.90) human tumor cell lines. Compound 5 showed higher cytotoxicity against NCI-H23 cells (non small lung cancer cell panel) as compared to standard drug vincristine sulphate. Further structural modification of these compounds may lead to potent anticancer activity.

A PCR-aided transcript titration assay (PATTY) to measure topoisomerase I gene expression in human tumor specimens.

PubMed

Meersma, G J; Bakker, M; Groen, H J; Van der Zee, A G; Jensen, P B; Giaccone, G; De Vries, E G; Smit, E F

1999-01-01

Topoisomerase I (topo I) inhibitors are promising anticancer agents with demonstrated activity against a wide range of solid tumors. Quantitative information on topol mRNA levels in tumor biopsies may predict response to topo I inhibitors. A polymerase chain reaction aided transcript titration assay (PATTY) was developed to allow quantitation of topol mRNA in small samples. Concentrations of topol mRNA in total RNA samples were estimated by RT-PCR analysis in a human small cell lung cancer (SCLC) cell line (GLC,) and its topotecan (GL2C/SK and F) and camptothecin (GL2C/Campt) resistant sublines, human non-small cell lung cancer (NSCLC) and ovarian carcinoma samples. Topol PATTY showed a decreased topo I mRNA level in GLC2/SK and F (4.5 pg/100 ng total RNA) and GLC,/Campt (2.2 pg/100 ng total RNA), respectively, compared to the parent cell line GLC2 (5.4 pg/100 ng total RNA). Topol protein levels as measured by Western blotting were compatible with topol mRNA levels. Median (range) topol mRNA levels were 3.23 (2.33-5.10) pg/100 ng total RNA in resected NSCLC specimen (n = 6), and 2.03 (0.54-0.95) pg/100 ng total RNA in resected ovarian cancer specimen (n = 6). We conclude that topol PATTY is a new assay that quantitates topol mRNA levels in cell lines and small tumor samples.

Autocrine activity of BDNF induced by the STAT3 signaling pathway causes prolonged TrkB activation and promotes human non-small-cell lung cancer proliferation

PubMed Central

Chen, Bo; Liang, Yan; He, Zheng; An, Yunhe; Zhao, Weihong; Wu, Jianqing

2016-01-01

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin superfamily, which has been implicated in the pathophysiology of the nervous system. Recently, several studies have suggested that BDNF and/or its receptor, tropomyosin related kinase B (TrkB), are involved in tumor growth and metastasis in several cancers, including prostate cancer, neuroblastoma, pancreatic ductal carcinoma, hepatocellular carcinoma, and lung cancer. Despite the increasing emphasis on BDNF/TrkB signaling in human tumors, how it participates in primary tumors has not yet been determined. Additionally, little is known about the molecular mechanisms that elicit signaling downstream of TrkB in the progression of non-small-cell lung cancer (NSCLC). In this study, we report the significant expression of BDNF in NSCLC samples and show that BDNF stimulation increases the synthesis of BDNF itself through activation of STAT3 in lung cancer cells. The release of BDNF can in turn activate TrkB signaling. The activation of both TrkB and STAT3 contribute to downstream signaling and promote human non-small-cell lung cancer proliferation. PMID:27456333

Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

PubMed

Qiu, Xiao-Xu; Liu, Yang; Zhang, Yi-Fan; Guan, Ya-Na; Jia, Qian-Qian; Wang, Chen; Liang, He; Li, Yong-Qin; Yang, Huang-Tian; Qin, Yong-Wen; Huang, Shuang; Zhao, Xian-Xian; Jing, Qing

2017-10-02

Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Identification of Novel Targets for Lung Cancer Therapy Using an Induced Pluripotent Stem Cell Model.

PubMed

Shukla, Vivek; Rao, Mahadev; Zhang, Hongen; Beers, Jeanette; Wangsa, Darawalee; Wangsa, Danny; Buishand, Floryne O; Wang, Yonghong; Yu, Zhiya; Stevenson, Holly; Reardon, Emily; McLoughlin, Kaitlin C; Kaufman, Andrew; Payabyab, Eden; Hong, Julie A; Zhang, Mary; Davis, Sean R; Edelman, Daniel C; Chen, Guokai; Miettinen, Markku; Restifo, Nicholas; Ried, Thomas; Meltzer, Paul S; Schrump, David S

2018-04-01

Despite extensive studies, the genetic and epigenetic mechanisms that mediate initiation and progression of lung cancers have not been fully elucidated. Previously, we have demonstrated that via complementary mechanisms, including DNA methylation, polycomb repressive complexes, and noncoding RNAs, cigarette smoke induces stem-like phenotypes that coincide with progression to malignancy in normal respiratory epithelia as well as enhanced growth and metastatic potential of lung cancer cells. To further investigate epigenetic mechanisms contributing to stemness/pluripotency in lung cancers and potentially identify novel therapeutic targets in these malignancies, induced pluripotent stem cells were generated from normal human small airway epithelial cells. Lung induced pluripotent stem cells were generated by lentiviral transduction of small airway epithelial cells of OSKM (Yamanaka) factors (octamer-binding transcription factor 4 [Oct4], sex-determining region Y box 2 [SOX2], Kruppel-like factor 4 [KLF4], and MYC proto-oncogene, bHLH transcription factor [MYC]). Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation sequencing analysis were performed. The lung induced pluripotent stem cells exhibited hallmarks of pluripotency, including morphology, surface antigen and stem cell gene expression, in vitro proliferation, and teratoma formation. In addition, lung induced pluripotent stem cells exhibited no chromosomal aberrations, complete silencing of reprogramming transgenes, genomic hypermethylation, upregulation of genes encoding components of polycomb repressive complex 2, hypermethylation of stem cell polycomb targets, and modulation of more than 15,000 other genes relative to parental small airway epithelial cells. Additional sex combs like-3 (ASXL3), encoding a polycomb repressive complex 2-associated protein not previously described in reprogrammed cells, was markedly upregulated in lung induced pluripotent stem cell as well as human small cell lung cancer lines and specimens. Overexpression of the additional sex combs like-3 gene correlated with increased genomic copy number in small cell lung cancer lines. Knock-down of the additional sex combs like-3 gene inhibited proliferation, clonogenicity, and teratoma formation by lung induced pluripotent stem cells and significantly diminished in vitro clonogenicity and growth of small cell lung cancer cells in vivo. Collectively, these studies highlight the potential utility of this lung induced pluripotent stem cell model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and suggest that additional sex combs like-3 is a novel target for small cell lung cancer therapy.

Bombesin-like peptide receptors in human bronchial epithelial cells.

PubMed

Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A

1996-01-01

Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

WNT-C59, a Small-Molecule WNT Inhibitor, Efficiently Induces Anterior Cortex That Includes Cortical Motor Neurons From Human Pluripotent Stem Cells.

PubMed

Motono, Makoto; Ioroi, Yoshihiko; Ogura, Takenori; Takahashi, Jun

2016-04-01

The recapitulation of human neural development in a controlled, defined manner from pluripotent stem cells (PSCs) has considerable potential for studies of human neural development, circuit formation and function, and the construction of in vitro models of neurological diseases. The inhibition of Wnt signaling, often by the recombinant protein DKK1, is important for the induction of cortical neurons. Here, we report a novel differentiation method using a small-molecule WNT inhibitor, WNT-C59 (C59), to efficiently induce human anterior cortex. We compared two types of small molecules, C59 and XAV939 (XAV), as substitutes for DKK1 to induce cortical neurons from PSCs in serum-free embryoid body-like aggregate culture. DKK1 and XAV inhibited only the canonical pathway of Wnt signaling, whereas C59 inhibited both the canonical and noncanonical pathways. C59 efficiently induced CTIP2+/COUP-TF1- cells, which are characteristic of the cells found in the anterior cortex. In addition, when grafted into the cortex of adult mice, the C59-induced cells showed abundant axonal fiber extension toward the spinal cord. These results raise the possibility of C59 contributing to cell replacement therapy for motor neuron diseases or insults. For a cell therapy against damaged corticospinal tract caused by neurodegenerative diseases or insults, cortical motor neurons are needed. Currently, their induction from pluripotent stem cells is considered very promising; however, an efficient protocol to induce motor neurons is not available. For efficient induction of anterior cortex, where motor neurons are located, various WNT inhibitors were investigated. It was found that one of them could induce anterior cortical cells efficiently. In addition, when grafted into the cortex of adult mice, the induced cells showed more abundant axonal fiber extension toward spinal cord. These results raise the possibility that this inhibitor contributes to a cell-replacement therapy for motor neuron diseases or insults. ©AlphaMed Press.

Small molecule AT7867 proliferates PDX1-expressing pancreatic progenitor cells derived from human pluripotent stem cells.

PubMed

Kimura, Azuma; Toyoda, Taro; Nishi, Yohei; Nasu, Makoto; Ohta, Akira; Osafune, Kenji

2017-10-01

While pancreatic islet transplantation achieves insulin independence in type 1 diabetes (T1D) patients, its widespread application is limited by donor tissue scarcity. Pancreatic progenitor cells (PPCs) give rise to all cell types in the pancreas during development. PPCs derived from human pluripotent stem cells have been shown to differentiate into functional β cells both in vitro and in vivo, and to reverse hyperglycemia, at least in mice. Therefore, PPCs have great potential to serve as an alternative cell source for cell therapy, and the identification of compounds that facilitate PPC proliferation could provide stable and large-scale pancreatic cell preparation systems in clinical settings. Here, we developed and performed cell-based screens to identify small molecules that induce the proliferation of hiPSC-derived PDX1-expressing PPCs. The screening identified AT7867, which promoted PPC proliferation approximately five-fold within six days through the maintenance of a high Ki67 + cell ratio. The induced proliferation by AT7867 does not result in DNA damage, as revealed by pHH2AX staining, and is observed specifically in PPCs but not other cell types. The established platform utilizing small molecules for PPC proliferation may contribute to the development of cell therapy for T1D using a regenerative medicine approach. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem and Progenitor Cells from Non‐Enriched Umbilical Cord Blood with Azole Compounds

PubMed Central

Bari, Sudipto; Zhong, Qixing; Fan, Xiubo; Poon, Zhiyong; Lim, Alvin Soon Tiong; Lim, Tse Hui; Dighe, Niraja; Li, Shang; Chiu, Gigi Ngar Chee; Chai, Christina Li Lin

2018-01-01

Abstract Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non‐enriched mononucleated cells (MNC) using novel azole‐based small molecules. Freshly‐thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure‐activity‐relationship studies of SB203580, a known p38‐MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8‐fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7‐fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600‐fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+CD34+ cells in the PB and BM by day 21 compared to non‐expanded and cytokine expanded grafts. The C7 expanded grafts maintained long‐term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre‐culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376–393 PMID:29392885

Epigenetic therapy potential of suberoylanilide hydroxamic acid on invasive human non-small cell lung cancer cells.

PubMed

Zhang, Shirong; Wu, Kan; Feng, Jianguo; Wu, Zhibing; Deng, Qinghua; Guo, Chao; Xia, Bing; Zhang, Jing; Huang, Haixiu; Zhu, Lucheng; Zhang, Ke; Shen, Binghui; Chen, Xufeng; Ma, Shenglin

2016-10-18

Metastasis is the reason for most cancer death, and a crucial primary step for cancer metastasis is invasion of the surrounding tissue, which may be initiated by some rare tumor cells that escape the heterogeneous primary tumor. In this study, we isolated invasive subpopulations of cancer cells from human non-small cell lung cancer (NSCLC) H460 and H1299 cell lines, and determined the gene expression profiles and the responses of these invasive cancer cells to treatments of ionizing radiation and chemotherapeutic agents. The subpopulation of highly invasive NSCLC cells showed epigenetic signatures of epithelial-mesenchymal transition, cancer cell stemness, increased DNA damage repair and cell survival signaling. We also investigated the epigenetic therapy potential of suberoylanilide hydroxamic acid (SAHA) on invasive cancer cells, and found that SAHA suppresses cancer cell invasiveness and sensitizes cancer cells to treatments of IR and chemotherapeutic agents. Our results provide guidelines for identification of metastatic predictors and for clinical management of NSCLC. This study also suggests a beneficial clinical potential of SAHA as a chemotherapeutic agent for NSCLC patients.

Identification of Small Peptides in Human Cerebrospinal Fluid upon Amyloid-β Degradation.

PubMed

Mizuta, Naoki; Yanagida, Kanta; Kodama, Takashi; Tomonaga, Takeshi; Takami, Mako; Oyama, Hiroshi; Kudo, Takashi; Ikeda, Manabu; Takeda, Masatoshi; Tagami, Shinji; Okochi, Masayasu

2017-01-01

Amyloid-β (Aβ) degradation in brains of Alzheimer disease patients is a crucial focus for the clarification of disease pathogenesis. Nevertheless, the mechanisms underlying Aβ degradation in the human brain remain unclear. This study aimed to quantify the levels of small C-terminal Aβ fragments generated upon Aβ degradation in human cerebrospinal fluid (CSF). A fraction containing small peptides was isolated and purified from human CSF by high-pressure liquid chromatography. Degradation products of Aβ C termini were identified and measured by liquid chromatography-tandem mass spectrometry. The C-terminal fragments of Aβ in the conditioned medium of cultured cells transfected with the Swedish variant of βAPP (sw βAPP) were analyzed. These fragments in brains of PS1 I213T knock-in transgenic mice, overexpressing sw βAPP, were also analyzed. The peptide fragments GGVV and GVV, produced by the cleavage of Aβ40, were identified in human CSF as well as in the brains of the transgenic mice and in the conditioned medium of the cultured cells. Relative to Aβ40 levels, GGVV and GVV levels were 7.6 ± 0.81 and 1.5 ± 0.18%, respectively, in human CSF. Levels of the GGVV fragment did not increase by the introduction of genes encoding neprilysin and insulin-degrading enzyme to the cultured cells. Our results indicate that a substantial amount of Aβ40 in human brains is degraded via a neprilysin- or insulin-degrading enzyme-independent pathway. © 2017 S. Karger AG, Basel.

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

PubMed

Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

2016-06-01

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Effect of HSP27 on Human Breast Tumor Cell Growth and Motility.

DTIC Science & Technology

1997-09-01

the small heat shock protein, Hsp27 , on growth and motility characteristics of human mammary tumor cell lines. Since Hsp27 regulates actin...microfilament dynamics, we hypothesize that cells expressing high levels of Hsp27 will show increased motility and altered chemotactic properties, in addition to...significantly elevated levels of Hsp27 has proven to be daunting. Down regulation of Hsp27 levels in MCF7 cells using antisense technology has also

Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

NASA Technical Reports Server (NTRS)

Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

2002-01-01

Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

A cell culture technique for human epiretinal membranes to describe cell behavior and membrane contraction in vitro.

PubMed

Wertheimer, Christian; Eibl-Lindner, Kirsten H; Compera, Denise; Kueres, Alexander; Wolf, Armin; Docheva, Denitsa; Priglinger, Siegfried G; Priglinger, Claudia; Schumann, Ricarda G

2017-11-01

To introduce a human cell culture technique for investigating in-vitro behavior of primary epiretinal cells and membrane contraction of fibrocellular tissue surgically removed from eyes with idiopathic macular pucker. Human epiretinal membranes were harvested from ten eyes with idiopathic macular pucker during standard vitrectomy. Specimens were fixed on cell culture plastic using small entomological pins to apply horizontal stress to the tissue, and then transferred to standard cell culture conditions. Cell behavior of 400 epiretinal cells from 10 epiretinal membranes was observed in time-lapse microscopy and analyzed in terms of cell migration, cell velocity, and membrane contraction. Immunocytochemistry was performed for cell type-specific antigens. Cell specific differences in migration behavior were observed comprising two phenotypes: (PT1) epiretinal cells moving fast, less directly, with small round phenotype and (PT2) epiretinal cells moving slowly, directly, with elongated large phenotype. No mitosis, no outgrowth and no migration onto the plastic were seen. Horizontal contraction measurements showed variation between specimens. Masses of epiretinal cells with a myofibroblast-like phenotype expressed cytoplasmatic α-SMA stress fibers and correlated with cell behavior characteristics (PT2). Fast moving epiretinal cells (PT1) were identified as microglia by immunostaining. This in-vitro technique using traction application allows for culturing surgically removed epiretinal membranes from eyes with idiopathic macular pucker, demonstrating cell behavior and membrane contraction of primary human epiretinal cells. Our findings emphasize the abundance of myofibroblasts, the presence of microglia and specific differences of cell behavior in these membranes. This technique has the potential to improve the understanding of pathologies at the vitreomacular interface and might be helpful in establishing anti-fibrotic treatment strategies.

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein.

PubMed

Eaton, A D; Zimmermann, C; Delaney, B; Hurley, B P

2017-08-01

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell ® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell ® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Human RNA polymerase II associated factor 1 complex promotes tumorigenesis by activating c-MYC transcription in non-small cell lung cancer

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

Zhi, Xiuyi; Giroux-Leprieur, Etienne; Respiratory Diseases and Thoracic Oncology Department, Ambroise Pare Hospital – APHP, Versailles Saint Quentin en Yvelines University, 9 Avenue Charles de Gaulle, 92100, Boulogne-Billancourt

2015-10-02

Human RNA polymerase II (RNAPII)-associated factor 1 complex (hPAF1C) plays a crucial role in protein-coding gene transcription. Overexpression of hPAF1C has been implicated in the initiation and progression of various human cancers. However, the molecular pathways involved in tumorigenesis through hPAF1C remain to be elucidated. The current study suggested hPAF1C expression as a prognostic biomarker for early stage non-small cell lung cancer (NSCLC) and patients with low hPAF1C expression levels had significantly better overall survival. Furthermore, the expression of hPAF1C was found to be positively correlated with c-MYC expression in patient tumor samples and in cancer cell lines. Mechanistic studiesmore » indicated that hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription. These results demonstrated the prognostic value of hPAF1C in early-stage NSCLC and the role of hPAF1C in the transcriptional regulation of c-MYC oncogene during NSCLC tumorigenesis. - Highlights: • hPAF1C expression is a prognostic biomarker for early stage non-small cell lung cancer. • The expression of hPAF1C was positively correlated with c-MYC in tumor samples of patients and in several NSCLC cell lines. • hPAF1C could promote lung cancer cell proliferation through regulating c-MYC transcription.« less

Cell biology and biotechnology research for exploration of the Moon and Mars

NASA Astrophysics Data System (ADS)

Pellis, N.; North, R.

Health risks generated by human long exposure to radiation, microgravity, and unknown factors in the planetary environment are the major unresolved issues for human space exploration. A complete characterization of human and other biological systems adaptation processes to long-duration space missions is necessary for the development of countermeasures. The utilization of cell and engineered tissue cultures in space research and exploration complements research in human, animal, and plant subjects. We can bring a small number of humans, animals, or plants to the ISS, Moon, and Mars. However, we can investigate millions of their cells during these missions. Furthermore, many experiments can not be performed on humans, e.g. radiation exposure, cardiac muscle. Cells from critical tissues and tissue constructs per se are excellent subjects for experiments that address underlying mechanisms important to countermeasures. The development of cell tissue engineered for replacement, implantation of biomaterial to induce tissue regeneration (e.g. absorbable collagen matrix for guiding tissue regeneration in periodontal surgery), and immunoisolation (e.g. biopolymer coating on transplanted tissues to ward off immunological rejection) are good examples of cell research and biotechnology applications. NASA Cell Biology and Biotechnology research include Bone/Muscle and Cardiovascular cell culture and tissue engineering; Environmental Health and Life Support Systems; Immune System; Radiation; Gravity Thresholds ; and Advanced Biotechnology Development to increase the understanding of animal and plant cell adaptive behavior when exposed to space, and to advance technologies that facilitates exploration. Cell systems can be used to investigate processes related to food, microbial proliferation, waste management, biofilms and biomaterials. The NASA Cell Science Program has the advantage of conducting research in microgravity based on significantly small resources, and the ability to conduct experiments in the early phase of the development of requirements for exploration. Supporting the NASA concept of stepping stones, we believe that ground based, International Space Station, robotic and satellite missions offer the ideal environment to perform experiments and secure answers necessary for human exploration.

Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment.

PubMed

Sakurai, Yasuteru; Kolokoltsov, Andrey A; Chen, Cheng-Chang; Tidwell, Michael W; Bauta, William E; Klugbauer, Norbert; Grimm, Christian; Wahl-Schott, Christian; Biel, Martin; Davey, Robert A

2015-02-27

Ebola virus causes sporadic outbreaks of lethal hemorrhagic fever in humans, but there is no currently approved therapy. Cells take up Ebola virus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebola virus entry into host cells requires the endosomal calcium channels called two-pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs, or small-molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule that we tested, inhibited infection of human macrophages, the primary target of Ebola virus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebola virus infection and may be effective targets for antiviral therapy. Copyright © 2015, American Association for the Advancement of Science.

Knockdown of human serine/threonine kinase 33 suppresses human small cell lung carcinoma by blocking RPS6/BAD signaling transduction.

PubMed

Sun, E L; Liu, C X; Ma, Z X; Mou, X Y; Mu, X A; Ni, Y H; Li, X L; Zhang, D; Ju, Y R

2017-01-01

Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.

Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer.

PubMed

Li, Chao; Ruan, Jing; Yang, Meng; Pan, Fei; Gao, Guo; Qu, Su; Shen, You-Lan; Dang, Yong-Jun; Wang, Kan; Jin, Wei-Lin; Cui, Da-Xiang

2015-09-01

Human induced pluripotent stem (iPS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human iPS cells labeled with fluorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Human iPS cells were prepared and cultured for 72 h. The culture medium was collected, and then was co-incubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human iPS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. iPS cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iPS cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer.

Phosphorylated Heat Shock Protein 20 (HSPB6) Regulates Transforming Growth Factor-α-Induced Migration and Invasion of Hepatocellular Carcinoma Cells.

PubMed

Matsushima-Nishiwaki, Rie; Toyoda, Hidenori; Nagasawa, Tomoaki; Yasuda, Eisuke; Chiba, Naokazu; Okuda, Seiji; Maeda, Atsuyuki; Kaneoka, Yuji; Kumada, Takashi; Kozawa, Osamu

2016-01-01

Human hepatocellular carcinoma (HCC) is one of the major malignancies in the world. Small heat shock proteins (HSPs) are reported to play an important role in the regulation of a variety of cancer cell functions, and the functions of small HSPs are regulated by post-translational modifications such as phosphorylation. We previously reported that protein levels of a small HSP, HSP20 (HSPB6), decrease in vascular invasion positive HCC compared with those in the negative vascular invasion. Therefore, in the present study, we investigated whether HSP20 is implicated in HCC cell migration and the invasion using human HCC-derived HuH7 cells. The transforming growth factor (TGF)-α-induced migration and invasion were suppressed in the wild-type-HSP20 overexpressed cells in which phosphorylated HSP20 was detected. Phospho-mimic-HSP20 overexpression reduced the migration and invasion compared with unphosphorylated HSP20 overexpression. Dibutyryl cAMP, which enhanced the phosphorylation of wild-type-HSP20, significantly reduced the TGF-α-induced cell migration of wild-type HSP20 overexpressed cells. The TGF-α-induced cell migration was inhibited by SP600125, a c-Jun N-terminal kinases (JNK) inhibitor. In phospho-mimic-HSP20 overexpressed HuH7 cells, TGF-α-stimulated JNK phosphorylation was suppressed compared with the unphosphorylated HSP20 overexpressed cells. Moreover, the level of phospho-HSP20 protein in human HCC tissues was significantly correlated with tumor invasion. Taken together, our findings strongly suggest that phosphorylated HSP20 inhibits TGF-α-induced HCC cell migration and invasion via suppression of the JNK signaling pathway.

A robust response to combination immune checkpoint inhibitor therapy in HPV-related small cell cancer: a case report.

PubMed

Ho, Won Jin; Rooper, Lisa; Sagorsky, Sarah; Kang, Hyunseok

2018-05-09

Human papillomavirus-related small cell carcinoma of the head and neck is an extremely rare, aggressive subtype with poor outcomes. Therapeutic options are limited and are largely adopted from small cell lung cancer treatment paradigms. This report describes a 69-year old male who was diagnosed of HPV-related oropharyngeal cancer with mixed small cell and squamous cell pathology which was clinically aggressive and progressed through multimodal platinum-based therapies. Upon manifestation of worsening metastatic disease, the patient was initiated on a combination of ipilimumab and nivolumab. Within 2 months of starting immunotherapy, a robust partial response was observed. During the treatment course, the patient developed immune-related adverse effects including new diabetes mellitus, colitis, and hypothyroidism. The disease-specific survival was 26 months. Combination immunotherapy may be an attractive option for HPV-related small cell head and neck cancers resistant to other treatment modalities and thus warrants further evaluation.

Oncogenic Ras: A double-edged sword for human epidermal stem and transient amplifying cells

PubMed Central

Dellambra, Elena

2016-01-01

ABSTRACT The human epidermal clonal evolution, i.e. the transition from stem cells (SCs) to transient amplifying (TA)-cells and post-mitotic cells, is a continuous and tightly regulated process that ensures physiologic tissue homeostasis. The Ras family of small GTPases has a key role in skin homeostasis and tumorigenesis. Indeed, activating mutations in Ras genes have been found in human cutaneous squamous cell carcinomas (cSCCs) and in experimentally-induced murine cSCCs. In mouse models, the Ras signaling might lead to hyperproliferative phenotypes, including the development of cSCCs, depending on the nature of the founding cells. Tumor-initiating cells or Cancer Stem Cells (CSCs) have been demonstrated in murine and human cSCCs even if the mechanism of their development from normal SCs or TA-cells is not completely elucidated. Here, the relation between the Ras expression outcome and the clonogenic potential of the target keratinocyte is discussed. PMID:27111451

Identification and isolation from either adult human bone marrow or G-CSF-mobilized peripheral blood of CD34(+)/CD133(+)/CXCR4(+)/ Lin(-)CD45(-) cells, featuring morphological, molecular, and phenotypic characteristics of very small embryonic-like (VSEL) stem cells.

PubMed

Sovalat, Hanna; Scrofani, Maurice; Eidenschenk, Antoinette; Pasquet, Stéphanie; Rimelen, Valérie; Hénon, Philippe

2011-04-01

Recently, we demonstrated that normal human bone marrow (hBM)-derived CD34(+) cells, released into the peripheral blood after granulocyte colony-stimulating factor mobilization, contain cell subpopulations committed along endothelial and cardiac differentiation pathways. These subpopulations could play a key role in the regeneration of post-ischemic myocardial lesion after their direct intracardiac delivery. We hypothesized that these relevant cells might be issued from very small embryonic-like stem cells deposited in the BM during ontogenesis and reside lifelong in the adult BM, and that they could be mobilized into peripheral blood by granulocyte colony-stimulating factor. Samples of normal hBM and leukapheresis products harvested from cancer patients after granulocyte colony-stimulating factor mobilization were analyzed and sorted by multiparameter flow cytometry strategy. Immunofluorescence and reverse transcription quantitative polymerase chain reaction assays were performed to analyze the expression of typical pluripotent stem cells markers. A population of CD34(+)/CD133(+)/CXCR4(+)/Lin(-) CD45(-) immature cells was first isolated from the hBM or from leukapheresis products. Among this population, very small (2-5 μm) cells expressing Oct-4, Nanog, and stage-specific embryonic antigen-4 at protein and messenger RNA levels were identified. Our study supports the hypothesis that very small embryonic-like stem cells constitute a "mobile" pool of primitive/pluripotent stem cells that could be released from the BM into the peripheral blood under the influence of various physiological or pathological stimuli. In order to fully support that hBM- and leukapheresis product-derived very small embryonic-like stem cells are actually pluripotent, we are currently testing their ability to differentiate in vitro into cells from all three germ layers. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Ubiquitin-proteasomal degradation of COX-2 in TGF-β stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I.

PubMed

Singh, Mohan; Chaudhry, Parvesh; Parent, Sophie; Asselin, Eric

2012-01-01

Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

Approaches to improve angiogenesis in tissue-engineered skin.

PubMed

Sahota, Parbinder S; Burn, J Lance; Brown, Nicola J; MacNeil, Sheila

2004-01-01

A problem with tissue-engineered skin is clinical failure due to delays in vascularization. The aim of this study was to explore a number of simple strategies to improve angiogenesis/vascularization using a tissue-engineered model of skin to which small vessel human dermal microvascular endothelial cells were added. For the majority of these studies, a modified Guirguis chamber was used, which allowed the investigation of several variables within the same experiment using the same human dermis; cell type, angiogenic growth factors, the influence of keratinocytes and fibroblasts, mechanical penetration of the human dermis, the site of endothelial cell addition, and the influence of hypoxia were all examined. A qualitative scoring system was used to assess the impact of these factors on the penetration of endothelial cells throughout the dermis. Similar results were achieved using freshly isolated small vessel human dermal microvascular endothelial cells or an endothelial cell line and a minimum cell seeding density was identified. Cell penetration was not influenced by the addition of angiogenic growth factors (vascular endothelial growth factor and basic fibroblast growth factor); similarly, including epidermal keratinocytes or dermal fibroblasts did not encourage endothelial cell entry, and neither did mechanical introduction of holes throughout the dermis. Two factors were identified that significantly enhanced endothelial cell penetration into the dermis: hypoxia and the site of endothelial cell addition. Endothelial cells added from the papillary surface entered into the dermis much more effectively than when cells were added to the reticular surface of the dermis. We conclude that this model is valuable in improving our understanding of how to enhance vascularization of tissue-engineered grafts.

A Life-Cycle Model of Human Social Groups Produces a U-Shaped Distribution in Group Size.

PubMed

Salali, Gul Deniz; Whitehouse, Harvey; Hochberg, Michael E

2015-01-01

One of the central puzzles in the study of sociocultural evolution is how and why transitions from small-scale human groups to large-scale, hierarchically more complex ones occurred. Here we develop a spatially explicit agent-based model as a first step towards understanding the ecological dynamics of small and large-scale human groups. By analogy with the interactions between single-celled and multicellular organisms, we build a theory of group lifecycles as an emergent property of single cell demographic and expansion behaviours. We find that once the transition from small-scale to large-scale groups occurs, a few large-scale groups continue expanding while small-scale groups gradually become scarcer, and large-scale groups become larger in size and fewer in number over time. Demographic and expansion behaviours of groups are largely influenced by the distribution and availability of resources. Our results conform to a pattern of human political change in which religions and nation states come to be represented by a few large units and many smaller ones. Future enhancements of the model should include decision-making rules and probabilities of fragmentation for large-scale societies. We suggest that the synthesis of population ecology and social evolution will generate increasingly plausible models of human group dynamics.

A Life-Cycle Model of Human Social Groups Produces a U-Shaped Distribution in Group Size

PubMed Central

Salali, Gul Deniz; Whitehouse, Harvey; Hochberg, Michael E.

2015-01-01

One of the central puzzles in the study of sociocultural evolution is how and why transitions from small-scale human groups to large-scale, hierarchically more complex ones occurred. Here we develop a spatially explicit agent-based model as a first step towards understanding the ecological dynamics of small and large-scale human groups. By analogy with the interactions between single-celled and multicellular organisms, we build a theory of group lifecycles as an emergent property of single cell demographic and expansion behaviours. We find that once the transition from small-scale to large-scale groups occurs, a few large-scale groups continue expanding while small-scale groups gradually become scarcer, and large-scale groups become larger in size and fewer in number over time. Demographic and expansion behaviours of groups are largely influenced by the distribution and availability of resources. Our results conform to a pattern of human political change in which religions and nation states come to be represented by a few large units and many smaller ones. Future enhancements of the model should include decision-making rules and probabilities of fragmentation for large-scale societies. We suggest that the synthesis of population ecology and social evolution will generate increasingly plausible models of human group dynamics. PMID:26381745

Sensitization of human carcinoma cells to alkylating agents by small interfering RNA suppression of 3-alkyladenine-DNA glycosylase.

PubMed

Paik, Johanna; Duncan, Tod; Lindahl, Tomas; Sedgwick, Barbara

2005-11-15

One of the major cytotoxic lesions generated by alkylating agents is DNA 3-alkyladenine, which can be excised by 3-alkyladenine DNA glycosylase (AAG). Inhibition of AAG may therefore result in increased cellular sensitivity to chemotherapeutic alkylating agents. To investigate this possibility, we have examined the role of AAG in protecting human tumor cells against such agents. Plasmids that express small interfering RNAs targeted to two different regions of AAG mRNA were transfected into HeLa cervical carcinoma cells and A2780-SCA ovarian carcinoma cells. Stable derivatives of both cell types with low AAG protein levels were sensitized to alkylating agents. Two HeLa cell lines with AAG protein levels reduced by at least 80% to 90% displayed a 5- to 10-fold increase in sensitivity to methyl methanesulfonate, N-methyl-N-nitrosourea, and the chemotherapeutic drugs temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea. These cells showed no increase in sensitivity to UV light or ionizing radiation. After treatment with methyl methanesulfonate, AAG knockdown HeLa cells were delayed in S phase but accumulated in G2-M. Our data support the hypothesis that ablation of AAG activity in human tumor cells may provide a useful strategy to enhance the efficacy of current chemotherapeutic regimens that include alkylating agents.

Sources and functions of extracellular small RNAs in human circulation

PubMed Central

Fritz, Joëlle V.; Heintz-Buschart, Anna; Ghosal, Anubrata; Wampach, Linda; Etheridge, Alton; Galas, David; Wilmes, Paul

2017-01-01

Various biotypes of endogenous small RNAs (sRNAs) have been detected in human circulation including microRNAs, tRNA, rRNA and yRNA fragments. These extracellular sRNAs (ex-sRNAs) are packaged and secreted by many different cell types. Ex-sRNAs exhibit differences in abundance in several disease contexts and have therefore been proposed as well-suited biomarkers. Furthermore, exosome-borne ex-sRNAs have been reported to elicit physiological responses in receiving cells. Albeit controversial, exogenous ex-sRNAs derived from plants and microorganisms have also been described in human blood. Essential questions which remain to be conclusively addressed in the field concern the (i) presence and mechanistic sources of exogenous ex-sRNA in human bodily fluids, (ii) detection and measurement of ex-sRNA in human circulation, (iii) selectivity of ex-sRNA export and import, (iv) sensitivity and specificity of ex-sRNA delivery to cellular targets, and (v) cell-, tissue-, organ- and organism-wide impacts of ex-sRNAs. We will survey the present state of knowledge of most of these questions in this review. PMID:27215587

MiR-21 suppresses the anticancer activities of curcumin by targeting PTEN gene in human non-small cell lung cancer A549 cells.

PubMed

Zhang, W; Bai, W; Zhang, W

2014-08-01

Curcumin, a natural phytochemical, exhibits potent anticancer activities. Here, we sought to determine the molecular mechanisms underlying the cytotoxic effects of curcumin against human non-small cell lung cancer (NSCLC) cells. MTT assay and annexin-V/PI staining were used to analyze the effects of curcumin on the proliferation and apoptosis of A549 cells. The expression of microRNA-21 in curcumin-treated A549 cells was measured by quantitative real-time polymerase chain reaction assay. The protein level of phosphatase and tensin homolog (PTEN), a putative target of microRNA-21, was determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA was performed to modulate the expression of microRNA-21 and PTEN under the treatment of curcumin. Curcumin at 20-40 μM inhibited cell proliferation and induced apoptosis in A549 cells. Curcumin treatment produced a dose-dependent and significant (P < 0.05) suppression of microRNA-21 expression, compared to untreated A549 cells. Moreover, the protein level of PTEN, a putative target of microRNA-21, was significantly elevated in curcumin-treated A549 cells, as determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA significantly (P < 0.05) reversed the growth suppression and apoptosis induction by curcumin, compared to corresponding controls. Our data suggest a novel molecular mechanism in which inhibition of microRNA-21 and upregulation of PTEN mediate the anticancer activities of curcumin in NSCLC cells. Suppression of microRNA-21 may thus have therapeutic benefits against this malignancy.

Effect of Wild-Type Shigella Species and Attenuated Shigella Vaccine Candidates on Small Intestinal Barrier Function, Antigen Trafficking, and Cytokine Release

PubMed Central

Fiorentino, Maria; Levine, Myron M.

2014-01-01

Bacterial dysentery due to Shigella species is a major cause of morbidity and mortality worldwide. The pathogenesis of Shigella is based on the bacteria's ability to invade and replicate within the colonic epithelium, resulting in severe intestinal inflammatory response and epithelial destruction. Although the mechanisms of pathogenesis of Shigella in the colon have been extensively studied, little is known on the effect of wild-type Shigella on the small intestine and the role of the host response in the development of the disease. Moreover, to the best of our knowledge no studies have described the effects of apically administered Shigella flexneri 2a and S. dysenteriae 1 vaccine strains on human small intestinal enterocytes. The aim of this study was to assess the coordinated functional and immunological human epithelial responses evoked by strains of Shigella and candidate vaccines on small intestinal enterocytes. To model the interactions of Shigella with the intestinal mucosa, we apically exposed monolayers of human intestinal Caco2 cells to increasing bacterial inocula. We monitored changes in paracellular permeability, examined the organization of tight-junctions and the pro-inflammatory response of epithelial cells. Shigella infection of Caco2 monolayers caused severe mucosal damage, apparent as a drastic increase in paracellular permeability and disruption of tight junctions at the cell-cell boundary. Secretion of pro-inflammatory IL-8 was independent of epithelial barrier dysfunction. Shigella vaccine strains elicited a pro-inflammatory response without affecting the intestinal barrier integrity. Our data show that wild-type Shigella infection causes a severe alteration of the barrier function of a small intestinal cell monolayer (a proxy for mucosa) and might contribute (along with enterotoxins) to the induction of watery diarrhea. Diarrhea may be a mechanism by which the host attempts to eliminate harmful bacteria and transport them from the small to the large intestine where they invade colonocytes inducing a strong inflammatory response. PMID:24416363

Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl.

PubMed

Yoshida, Hiroshi; Yamada, Hajime; Nogami, Wataru; Dohi, Keiji; Kurino-Yamada, Tomomi; Sugiyama, Koji; Takahashi, Koji; Gahara, Yoshinari; Kitaura, Motoji; Hasegawa, Minoru; Oshima, Itsuki; Kuwabara, Kenji

2018-03-01

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production. Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Adenovirus small interfering RNA targeting ezrin induces apoptosis and inhibits metastasis of human osteosarcoma MG-63 cells.

PubMed

Tao, Zhi-Wei; Zou, Ping-An

2018-06-13

Osteosarcoma is a disease prone to recurrence and metastasis, and adenovirus expression vector is frequently studied as a therapeutic target of osteosarcoma in recent year. This study attempts to explore the effect of adenovirus-mediated small interfering RNA (siRNA) targeting ezrin on the proliferation, migration, invasion and apoptosis of human osteosarcoma MG-63 cells. Human osteosarcoma MG-63 cell line was selected for construction of recombinant adenovirus vector. The mRNA and protein levels of ezrin, Bcl2-associated X protein (Bax), B cell lymphoma-2 (Bcl-2), p21, p53, Caspase-3, matrix metalloproteinase 2 (MMP-2) and MMP-9, Cyclin D1, and cyclin-dependent kinase 4a (CDK4a) were determined. Through ELISA, the levels of Caspase-3, MMP-2 and MMP-9 were examined. Finally, human osteosarcoma MG-63 cell viability, growth, invasion, migration, and apoptosis were detected. Initially, adenovirus expression vector of ezrin was constructed by ezrin 2 siRNA sequence. Adenovirus-mediated siRNA targeting ezrin reduced expression of ezrin in MG-63 cells. The results revealed that adenovirus-mediated siRNA targeting ezrin elevated expression levels of Bax, P21, P53, and Caspase-3, Cyclin D1, and CDK4a and reduced expression levels of Bcl-2, MMP-2, and MMP-9. Furthermore, adenovirus-mediated siRNA targeting ezrin inhibited human osteosarcoma MG-63 cell viability, growth, invasion, and migration, and promoted apoptosis. Our study demonstrates that adenovirus-mediated siRNA targeting ezrin can induce apoptosis and inhibit the proliferation, migration and invasion of human osteosarcoma MG-63 cells. ©2018 The Author(s).

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine.

PubMed

Muglia, C; Mercer, N; Toscano, M A; Schattner, M; Pozner, R; Cerliani, J P; Gobbi, R Papa; Rabinovich, G A; Docena, G H

2011-05-26

Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals1(-/-)) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals1(-/-) mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.

Macrophages and Dendritic Cells Are the Predominant Cells Infected in Measles in Humans.

PubMed

Allen, Ingrid V; McQuaid, Stephen; Penalva, Rosana; Ludlow, Martin; Duprex, W Paul; Rima, Bertus K

2018-01-01

Characterization of human measles cases is essential in order to better assess the data generated in model systems of morbillivirus infection. To this end, we collected formalin-fixed tissue samples from 23 natural measles cases from different areas in the world and different phases of disease ranging from prodromal and acute measles to a persistent infection in an immunocompromised subject. We show that the vast majority of measles virus (MV)-infected cells in epithelia were intraepithelial immune cells that were, in most cases, positive for the CD11c myeloid cell marker. Small numbers of measles virus-infected cytokeratin-positive epithelial cells were also detected in bronchial and appendix epithelia. Dissolution and disruption of uninfected and MV-infected alveolar and bronchial epithelia were prominent features of the measles cases, especially in the established and late phases of the disease. In some instances, this was associated with the formation of MV-infected multinucleated giant cells which expressed CD11c and/or macrophage cell marker 68, a pathological feature also prominently observed in closely associated mucosa-associated lymphoid tissue. Collectively, these data show that resident and inflammatory infiltrating immune cells alter the architecture of respiratory tract epithelia and highlight the necessity for additional research into the function(s) and expression of nectin-4 in human tissues. IMPORTANCE We have brought together a unique collection of 23 human cases of measles infection and studied the types of cells that are infected. This work has not been done with modern technologies such as double labeling with antibodies and confocal microscopy in human cases primarily due to the fact that it is difficult to obtain the material because, fortunately, measles is fatal in only a very small fraction of infected patients. During the past decades, the receptors for measles virus have been elucidated and monkey models have been developed. We found that, in most cases, independently of whether the tissues were obtained early or later in the infection, the primary cell types that were infected were those of the immune system such as lymphocytes, macrophages, and dendritic cells. A very small number of epithelial cells were also found to be infected. Copyright © 2018 Allen et al.

Novel methods of treating ovarian infertility in older and POF women, testicular infertility, and other human functional diseases.

PubMed

Bukovsky, Antonin

2015-02-25

In vitro maturation (IVM) and in vitro fertilization (IVF) technologies are facing with growing demands of older women to conceive. Although ovarian stem cells (OSCs) of older women are capable of producing in vitro fresh oocyte-like cells (OLCs), such cells cannot respond to IVM and IVF due to the lack of granulosa cells required for their maturation. Follicular renewal is also dependent on support of circulating blood mononuclear cells. They induce intermediary stages of meiosis (metaphase I chromosomal duplication and crossover, anaphase, telophase, and cytokinesis) in newly emerging ovarian germ cells, as for the first time demonstrated here, induce formation of granulosa cells, and stimulate follicular growth and development. A pretreatment of OSC culture with mononuclear cells collected from blood of a young healthy fertile woman may cause differentiation of bipotential OSCs into both developing germ and granulosa cells. A small blood volume replacement may enable treatment of ovarian infertility in vivo. The transferred mononuclear cells may temporarily rejuvenate virtually all tissues, including improvement of the function of endocrine tissues. Formation of new follicles and their development may be sufficient for IVM and IVF. The novel proposed in vitro approaches may be used as a second possibility. Infertility of human males affects almost a half of the infertility cases worldwide. Small blood volume replacement from young healthy fertile men may also be easy approach for the improvement of sperm quality in older or other affected men. In addition, body rejuvenation by small blood volume replacement from young healthy individuals of the same sex could represent a decline of in vitro methodology in favor of in vivo treatment for human functional diseases. Here we propose for the first time that blood mononuclear cells are essential for rejuvenation of those tissues, where immune system components participate in an appropriate division and differentiation of tissue stem cells. If needed, small blood volume replacement from distinct young healthy individuals could be utilized in six month intervals for repair of young altered or aged reproductive and other tissue functions. Systemic and local use of honey bee propolis tincture is an alternative option for functional rejuvenation of some tissues.

Vasoactive Intestinal Peptide Inhibits Human Small-Cell Lung Cancer Proliferation in vitro and in vivo

NASA Astrophysics Data System (ADS)

Maruno, Kaname; Absood, Afaf; Said, Sami I.

1998-11-01

Small-cell lung carcinoma (SCLC) is an aggressive, rapidly growing and metastasizing, and highly fatal neoplasm. We report that vasoactive intestinal peptide inhibits the proliferation of SCLC cells in culture and dramatically suppresses the growth of SCLC tumor-cell implants in athymic nude mice. In both cases, the inhibition was mediated apparently by a cAMP-dependent mechanism, because the inhibition was enhanced by the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in proportion to increases in intracellular cAMP levels, and the inhibition was abolished by selective inhibition of cAMP-dependent protein kinase. If confirmed in clinical trials, this antiproliferative action of vasoactive intestinal peptide may offer a new and promising means of suppressing SCLC in human subjects, without the toxic side effects of chemotherapeutic agents.

Inhibition of West Nile Virus replication by retrovirus-delivered small interfering RNA in human neuroblastoma cells.

PubMed

Yang, Yongbo; Wu, Chengxiang; Wu, Jianguo; Nerurkar, Vivek R; Yanagihara, Richard; Lu, Yuanan

2008-05-01

West Nile virus (WNV) has been responsible for the largest outbreaks of arboviral encephalitis in U.S. history. No specific drug is currently available for the effective treatment of WNV infection. To exploit RNA interference as a potential therapeutic approach, a Moloney murine leukemia virus-based retrovirus vector was used to effectively deliver WNV-specific small interfering RNA (siRNA) into human neuroblastoma HTB-11 cells. Viral plaque assays demonstrated that transduced cells were significantly refractory to WNV replication, as compared to untransduced control cells (P < 0.05), which correlated with the reduced expression of target viral genes and respective viral proteins. Therefore, retrovirus-mediated delivery of siRNA for gene silencing can be used to study the specific functions of viral genes associated with replication and may have potential therapeutic applications.

Detection of PIWI and piRNAs in the mitochondria of mammalian cancer cells

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

Kwon, ChangHyuk, E-mail: netbuyer@hanmail.net; Tak, Hyosun, E-mail: chuberry@naver.com; Rho, Mina, E-mail: minarho@hanyang.ac.kr

2014-03-28

Highlights: • piRNA sequences were mapped to human mitochondrial (mt) genome. • We inspected small RNA-Seq datasets from somatic cell mt subcellular fractions. • Piwi and piRNA transcripts are present in mammalian somatic cancer cell mt fractions. - Abstract: Piwi-interacting RNAs (piRNAs) are 26–31 nt small noncoding RNAs that are processed from their longer precursor transcripts by Piwi proteins. Localization of Piwi and piRNA has been reported mostly in nucleus and cytoplasm of higher eukaryotes germ-line cells, where it is believed that known piRNA sequences are located in repeat regions of nuclear genome in germ-line cells. However, localization of PIWImore » and piRNA in mammalian somatic cell mitochondria yet remains largely unknown. We identified 29 piRNA sequence alignments from various regions of the human mitochondrial genome. Twelve out 29 piRNA sequences matched stem-loop fragment sequences of seven distinct tRNAs. We observed their actual expression in mitochondria subcellular fractions by inspecting mitochondrial-specific small RNA-Seq datasets. Of interest, the majority of the 29 piRNAs overlapped with multiple longer transcripts (expressed sequence tags) that are unique to the human mitochondrial genome. The presence of mature piRNAs in mitochondria was detected by qRT-PCR of mitochondrial subcellular RNAs. Further validation showed detection of Piwi by colocalization using anti-Piwil1 and mitochondria organelle-specific protein antibodies.« less

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer

PubMed Central

Weiskopf, Kipp; Jahchan, Nadine S.; Schnorr, Peter J.; Ring, Aaron M.; Maute, Roy L.; Volkmer, Anne K.; Volkmer, Jens-Peter; Liu, Jie; Lim, Jing Shan; Yang, Dian; Seitz, Garrett; Nguyen, Thuyen; Wu, Di; Guerston, Heather; Trapani, Francesca; George, Julie; Poirier, John T.; Gardner, Eric E.; Miles, Linde A.; de Stanchina, Elisa; Lofgren, Shane M.; Vogel, Hannes; Winslow, Monte M.; Dive, Caroline; Thomas, Roman K.; Rudin, Charles M.; van de Rijn, Matt; Majeti, Ravindra; Garcia, K. Christopher; Weissman, Irving L.

2016-01-01

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers. PMID:27294525

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

PubMed Central

Mello, Paola de Andrade; Filippi-Chiela, Eduardo Cremonese; Nascimento, Jéssica; Beckenkamp, Aline; Santana, Danielle Bertodo; Kipper, Franciele; Casali, Emerson André; Nejar Bruno, Alessandra; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Wink, Marcia Rosângela; Lenz, Guido; Buffon, Andréia

2014-01-01

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. PMID:25103241

Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation.

PubMed

Sharma, Arun; Li, Guang; Rajarajan, Kuppusamy; Hamaguchi, Ryoko; Burridge, Paul W; Wu, Sean M

2015-03-18

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

E3 ubiquitin ligase Pirh2 enhances tumorigenic properties of human non-small cell lung carcinoma cells

PubMed Central

Fedorova, Olga; Shuvalov, Oleg; Merkulov, Valeriy; Vasileva, Elena; Antonov, Alexey; Barlev, Nikolai A.

2016-01-01

The product of RCHY1 human gene, Pirh2, is a RING-finger containing E3 ligase that modifies p53 with ubiquitin residues resulting in its subsequent degradation in proteasomes. Transcription of RCHY1 is regulated by p53 itself thus forming a negative regulatory feedback loop. Functionally, by eliminating p53, Pirh2 facilitates tumorigenesis. However, the role of Pirh2 in cancer cells lacking p53 is yet not well understood. Therefore, we decided to elucidate the role of Pirh2 in p53-negative human non-small cell lung carcinoma cells, H1299. We found that ectopic expression of Pirh2 enhanced cell proliferation, resistance to doxorubicin, and increased migration potential. Ablation of Pirh2 by specific shRNA reversed these phenotypes. Mechanistically, Pirh2 increased mRNA and protein levels of the c-Myc oncogene. The bioinformatics data indicate that co-expression of both c-Myc and Pirh2 strongly correlated with poor survival of lung cancer patients. Collectively, our results suggest that Pirh2 can be considered as a potential pharmacological target for developing anticancer therapies to treat p53-negative cancers. PMID:28191284

Pharmacologic Induction of CD8+ T Cell Memory: Better Living Through Chemistry

PubMed Central

Gattinoni, Luca; Klebanoff, Christopher A.; Restifo, Nicholas P.

2011-01-01

The generation of a robust population of memory T cells is critical for effective vaccine and cell-based therapies to prevent and treat infectious diseases and cancer. A series of recent papers have established a new, cell-intrinsic approach in which small molecules target key metabolic and developmental pathways to enhance the formation and maintenance of highly functional CD8+ memory T cells. These findings raise the exciting new possibility of using small molecules, many of which are already approved for human use, for the pharmacologic induction of immunologic memory. PMID:20371454

Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer

PubMed Central

2012-01-01

Background G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. Methods The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. Results Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. Conclusion The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression. PMID:23273253

BH3-mimetic small molecule inhibits the growth and recurrence of adenoid cystic carcinoma

PubMed Central

Acasigua, Gerson A.; Warner, Kristy A.; Nör, Felipe; Helman, Joseph; Pearson, Alexander T.; Fossati, Anna C.; Wang, Shaomeng; Nör, Jacques E.

2015-01-01

Objectives To evaluate the anti-tumor effect of BM-1197, a new potent and highly specific small molecule inhibitor of Bcl-2/Bcl-xL, in preclinical models of human adenoid cystic carcinoma (ACC). Methods Low passage primary human adenoid cystic carcinoma cells (UM-HACC-2A,-2B,-5,-6) and patient-derived xenograft (PDX) models (UM-PDX-HACC) were developed from surgical specimens obtained from 4 patients. The effect of BM-1197 on cell viability and cell cycle were evaluated in vitro using this panel of low passage ACC cells. The effect of BM-1197 on tumor growth, recurrence and tumor cell apoptosis in vivo was evaluated with the PDX model of ACC (UM-PDX-HACC-5). Results Exposure of low passage primary human ACC cells to BM-1197 mediated an IC50 of 0.92-2.82 μM. This correlated with an increase in the fraction of apoptotic cells (p<0.0001) and an increase in caspase-3 activity (p<0.0001), but no noticeable differences in cell cycle (p>0.05). In vivo, BM-1197 inhibited tumor growth (p=0.0256) and induced tumor cell apoptosis (p=0.0165) without causing significant systemic toxicities, as determined by mouse weight over time. Surprisingly, weekly BM-1197 decreased the incidence of tumor recurrence (p=0.0297), as determined by Kaplan-Meier analysis. Conclusion These data demonstrated that single agent BM-1197 induces apoptosis and inhibits tumor growth in preclinical models of adenoid cystic carcinoma. Notably, single agent BM-1197 inhibited tumor recurrence, which is considered a major clinical challenge in the clinical management of adenoid cystic carcinoma. Collectively, these results suggest that patients with adenoid cystic carcinoma might benefit from therapy with a BH3-mimetic small molecule. PMID:26121939

Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer.

PubMed

Jala, Venkatakrishna Rao; Radde, Brandie N; Haribabu, Bodduluri; Klinge, Carolyn M

2012-12-28

G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression.

Human spleen and red blood cells

NASA Astrophysics Data System (ADS)

Pivkin, Igor; Peng, Zhangli; Karniadakis, George; Buffet, Pierre; Dao, Ming

2016-11-01

Spleen plays multiple roles in the human body. Among them is removal of old and altered red blood cells (RBCs), which is done by filtering cells through the endothelial slits, small micron-sized openings. There is currently no experimental technique available that allows us to observe RBC passage through the slits. It was previously noticed that people without a spleen have less deformable red blood cells, indicating that the spleen may play a role in defining the size and shape of red blood cells. We used detailed RBC model implemented within the Dissipative Particle Dynamics (DPD) simulation framework to study the filter function of the spleen. Our results demonstrate that spleen indeed plays major role in defining the size and shape of the healthy human red blood cells.

2011 Rita Schaffer lecture: nanoparticles for intracellular nucleic acid delivery.

PubMed

Green, Jordan J

2012-07-01

Nanoparticles are a promising technology for delivery of new types of therapeutics. A polymer library approach has allowed engineering of polymeric particles that are particularly effective for the delivery of DNA and siRNA to human cells. Certain chemical structural motifs, degradable linkages, hydrophobicity, and biophysical properties are key for successful intracellular delivery. Small differences to biomaterial structure, and especially the type of degradable linkage in the polymers, can be critical for successful delivery of siRNA vs. DNA. Furthermore, subtle changes to biomaterial structure can facilitate cell-type gene delivery specificity between human brain cancer cells and healthy cells as well as between human retinal endothelial cells and epithelial cells. These polymeric nanoparticles are effective for nucleic acid delivery in a broad range of human cell types and have applications to regenerative medicine, ophthalmology, and cancer among many other biomedical research areas.

The anti-human immunodeficiency virus agent 3'-fluorothymidine induces DNA damage and apoptosis in human lymphoblastoid cells.

PubMed Central

Sundseth, R; Joyner, S S; Moore, J T; Dornsife, R E; Dev, I K

1996-01-01

Patients infected with the human immunodeficiency virus experienced severe hematopoietic toxicity after treatment with the deoxynucleoside analog 3'-fluorothymidine (FLT). Using several methods for the analysis of genome integrity, including histochemical staining of the 3' ends of DNA and both conventional and pulsed-field agarose gel electrophoresis, we demonstrated that FLT caused extensive DNA fragmentation in CEM cells that was not observed when these cells were treated with other, less toxic thymidine analogs. In addition, a distinctive pattern of small DNA fragments that is characteristic of cells in the process of programmed cell death was observed in the genomic DNA of CEM cells treated with FLT. We conclude that FLT induces DNA fragmentation and apoptosis in a human cell line of hematopoietic origin, and we offer this observation as a possible explanation for the severe toxicity of FLT observed in vivo. PMID:8834875

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2

PubMed Central

Majumder, Anurima; Govindasamy, Lakshmanan; Magis, Andrew; Kiss, Róbert; Polgár, Tímea; Baskin, Rebekah; Allan, Robert W.; Agbandje-McKenna, Mavis; Reuther, Gary W.; Keserű, György M.; Bisht, Kirpal S.; Sayeski, Peter P.

2010-01-01

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential. PMID:20667821

Combination Therapy Targeting BCL6 and Phospho-STAT3 Defeats Intratumor Heterogeneity in a Subset of Non-Small Cell Lung Cancers. | Office of Cancer Genomics

Cancer.gov

Oncogene-specific changes in cellular signaling have been widely observed in lung cancer. Here, we investigated how these alterations could affect signaling heterogeneity and suggest novel therapeutic strategies. We compared signaling changes across six human bronchial epithelial cell (HBEC) strains that were systematically transformed with various combinations of TP53, KRAS, and MYC-oncogenic alterations commonly found in non-small cell lung cancer (NSCLC).

Human Lung Small Airway-on-a-Chip Protocol.

PubMed

Benam, Kambez H; Mazur, Marc; Choe, Youngjae; Ferrante, Thomas C; Novak, Richard; Ingber, Donald E

2017-01-01

Organs-on-chips are microfluidic cell culture devices created using microchip manufacturing techniques that contain hollow microchannels lined by living cells, which recreate specialized tissue-tissue interfaces, physical microenvironments, and vascular perfusion necessary to recapitulate organ-level physiology in vitro. Here we describe a protocol for fabrication, culture, and operation of a human lung "small airway-on-a-chip," which contains a differentiated, mucociliary bronchiolar epithelium exposed to air and an underlying microvascular endothelium that experiences fluid flow. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin rigid porous membrane; this requires less than 1 day to complete. Next, primary human airway bronchiolar epithelial cells isolated from healthy normal donors or patients with respiratory disease are cultured on the porous membrane within one microchannel while lung microvascular endothelial cells are cultured on the opposite side of the same membrane in the second channel to create a mucociliated epithelium-endothelium interface; this process take about 4-6 weeks to complete. Finally, culture medium containing neutrophils isolated from fresh whole human blood are flowed through the microvascular channel of the device to enable real-time analysis of capture and recruitment of circulating leukocytes by endothelium under physiological shear; this step requires less than 1 day to complete. The small airway-on-a-chip represents a new microfluidic tool to model complex and dynamic inflammatory responses of healthy and diseased lungs in vitro.

Analysis of the MDM2 antagonist nutlin-3 in human prostate cancer cells.

PubMed

Logan, Ian R; McNeill, Hesta V; Cook, Susan; Lu, Xiaohong; Lunec, John; Robson, Craig N

2007-06-01

Small molecule MDM2 antagonists including nutlin-3 have been shown to be effective against a range of cancer cell types and nutlin-3 can inhibit growth of LNCaP xenografts. We compared the efficacy of nutlin-3 in three prostate cancer cell types and provide an insight into the mechanism of nutlin-3. Nutlin-3 efficacy was measured using proliferation assays, cell cycle analysis, apoptosis assays, quantitative RT-PCR, and immunoblotting. Chromatin immunoprecipitation (ChIP) assays were also performed. Nutlin-3 can specifically inhibit proliferation of LNCaP cells through cell cycle arrest and apoptosis. This coincides with increased levels of the p53-responsive transcripts p21, PUMA, gadd45, and Mdm2 and recruitment of p53 to chromatin. Nutlin-3 also reduces androgen receptor levels, resulting in altered receptor recruitment to chromatin. Our study demonstrates that small molecule MDM2 antagonists might be useful in the treatment of human prostate cancers that retain functional p53 and androgen receptor signaling. Copyright 2007 Wiley-Liss, Inc.

A drug repositioning approach identifies tricyclic antidepressants as inhibitors of small cell lung cancer and other neuroendocrine tumors

PubMed Central

Jahchan, Nadine S; Dudley, Joel T; Mazur, Pawel K; Flores, Natasha; Yang, Dian; Palmerton, Alec; Zmoos, Anne-Flore; Vaka, Dedeepya; Tran, Kim QT; Zhou, Margaret; Krasinska, Karolina; Riess, Jonathan W; Neal, Joel W; Khatri, Purvesh; Park, Kwon S; Butte, Atul J; Sage, Julien

2013-01-01

Small cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with high mortality. We used a systematic drug-repositioning bioinformatics approach querying a large compendium of gene expression profiles to identify candidate FDA-approved drugs to treat SCLC. We found that tricyclic antidepressants and related molecules potently induce apoptosis in both chemonaïve and chemoresistant SCLC cells in culture, in mouse and human SCLC tumors transplanted into immunocompromised mice, and in endogenous tumors from a mouse model for human SCLC. The candidate drugs activate stress pathways and induce cell death in SCLC cells, at least in part by disrupting autocrine survival signals involving neurotransmitters and their G protein-coupled receptors. The candidate drugs inhibit the growth of other neuroendocrine tumors, including pancreatic neuroendocrine tumors and Merkel cell carcinoma. These experiments identify novel targeted strategies that can be rapidly evaluated in patients with neuroendocrine tumors through the repurposing of approved drugs. PMID:24078773

Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate Non-Human Primate Hearts

PubMed Central

Chong, James J.H.; Yang, Xiulan; Don, Creighton W.; Minami, Elina; Liu, Yen-Wen; Weyers, Jill J; Mahoney, William M.; Van Biber, Benjamin; Cook, Savannah M.; Palpant, Nathan J; Gantz, Jay; Fugate, James A.; Muskheli, Veronica; Gough, G. Michael; Vogel, Keith W.; Astley, Cliff A.; Hotchkiss, Charlotte E.; Baldessari, Audrey; Pabon, Lil; Reinecke, Hans; Gill, Edward A.; Nelson, Veronica; Kiem, Hans-Peter; Laflamme, Michael A.; Murry, Charles E.

2014-01-01

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure 1 by providing human cardiomyocytes to support heart regeneration 2. Studies of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in small animal models have shown favorable effects of this treatment 3–7. It remains unknown, however, whether clinical scale hESC-CMs transplantation is feasible, safe or can provide large-scale myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (>1 billion cells/batch) and cryopreserved with good viability. Using a non-human primate (NHP) model of myocardial ischemia-reperfusion, we show that that cryopreservation and intra-myocardial delivery of 1 billion hESC-CMs generates significant remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a three-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small animal models 7, non-fatal ventricular arrhythmias were observed in hESC-CM engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome. PMID:24776797

Kindlin-2 Expression in Arsenite and Cadmium Transformed Bladder Cancer Cell Lines and in Archival Specimens of Human Bladder Cancer

PubMed Central

Talaat, Sherine; Somji, Seema; Toni, Conrad; Garrett, Scott H.; Zhou, Xu Dong; Sens, Mary Ann; Sens, Donald A.

2011-01-01

Objective The goal of this study was to confirm a microarray study that suggested that Kindlin-2 might play a role in the development and progression of bladder cancer. There has been no previous examination of Kindlin-2 expression in human bladder cancer. Methods A combination of real time PCR, western analysis and immunohistochemistry was used to characterize Kindlin-2 expression in arsenite (As+3) and cadmium (Cd+2) transformed human cell lines, their tumor transplants in immune-compromised mice, and in archival specimens of human bladder and bladder cancer. Results The results show that the Kindlin-2 expression patterns in the cell lines were not duplicated in the tumor tissues. However, it was shown that Kindlin-2 was expressed in the stromal element of all the transplanted tumors and archival specimens of human bladder cancer. It was also shown that a small number of high grade invasive urothelial cancers have focal expression of Kindlin-2 in the tumor cells. Conclusion Kindlin-2 is expressed in the stromal component of most, if not all, human bladder cancers. Kindlin-2 is not expressed in normal urothelium. Kindlin-2 is expressed in a small subset of high grade invasive bladder cancers and may have potential as a prognostic marker for tumor progression. PMID:21624607

Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells.

PubMed

Yang, Tingting; Zhang, Hai-Liang; Liang, Qingnan; Shi, Yingtang; Mei, Yan-Ai; Barrett, Paula Q; Hu, Changlong

2016-09-01

Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism. © 2016 American Heart Association, Inc.

Ro small cytoplasmic ribonucleoproteins are a subclass of La ribonucleoproteins: further characterization of the Ro and La small ribonucleoproteins from uninfected mammalian cells.

PubMed Central

Hendrick, J P; Wolin, S L; Rinke, J; Lerner, M R; Steitz, J A

1981-01-01

Small ribonucleic acid (RNA)-protein complexes precipitated by anti-Ro and anti-La antibodies from lupus patients have been examined with emphasis on their RNA components. In both ribonucleoprotein (RNP) classes, the numbers of different RNA molecules and their sequences vary between mouse and human cells. The complex mixtures of La RNAs include two previously sequenced 4.5S RNAs from mouse cells and 5S ribosomal RNA-like molecules from both mouse and human cells. All Ro and La RNAs possess 5-triphosphates. Some La RNAs have internal modifications typical of transfer RNAs. The Ro RNPs are quite stable and are localized by immunofluorescence in the cell cytoplasm, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus. Despite these differences, reconstitution experiments show that the Ro particles carry the La as well as the Ro determinant. Studies using a nuclear transcription system demonstrate that most of the La RNAs are synthesized by RNA polymerase III. The possibility that the La protein(s) functions in the transcription or maturation of all RNA polymerase III transcripts is discussed. Images PMID:6180298

TC-1 Overexpression Promotes Cell Proliferation in Human Non-Small Cell Lung Cancer that Can Be Inhibited by PD173074

PubMed Central

Zhang, Na; Bai, Guangzhen; Zhong, Daixing; Su, Kai; Liu, Boya; Li, Xiaofei; Wang, Yunjie; Wang, Xiaoping

2014-01-01

Thyroid cancer-1 (TC-1), a natively disordered protein, is widely expressed in vertebrates and overexpressed in many kinds of tumors. However, its exact role and regulation mechanism in human non-small cell lung cancer (NSCLC) are still unclear. In the present study, we found that TC-1 is highly expressed in NSCLC and that its aberrant expression is strongly associated with NSCLC cell proliferation. Exogenous TC-1 overexpression promotes cell proliferation, accelerates the cell G1-to-S-phase transition, and reduces apoptosis in NSCLC. The knockdown of TC-1, however, inhibits NSCLC cell proliferation, cycle transition, and apoptosis resistance. Furthermore, we also demonstrated that PD173074, which functions as an inhibitor of the TC-1 in NSCLC, decreases the expression of TC-1 and inhibits TC-1 overexpression mediated cell proliferation in vitro and in vivo. Nevertheless, the inhibition function of PD173074 on NSCLC cell proliferation was eliminated in cells with TC-1 knockdown. These results suggest that PD173074 plays a significant role in TC-1 overexpression mediated NSCLC cell proliferation and may be a potential intervention target for the prevention of cell proliferation in NSCLC. PMID:24941347

Molecular evidence of viral DNA in non-small cell lung cancer and non-neoplastic lung

DOE PAGES

Robinson, Lary A.; Jaing, Crystal J.; Campbell, Christine Pierce; ...

2016-07-14

Although ~20% of human cancers are caused by microorganisms, only suspicion exists for a microbial cause of lung cancer. Potential infectious agents were investigated in non-small cell lung cancer (NSCLC) and non-neoplastic lung. Seventy NSCLC tumours (33 squamous cell carcinomas, 17 adenocarcinomas, 10 adenocarcinomas with lepidic spread, and 10 oligometastases) and 10 non-neoplastic lung specimens were evaluated for molecular evidence of microorganisms. Tissues were subjected to the Lawrence Livermore Microbial Detection Array, an oncovirus panel of the International Agency for Research on Cancer, and human papillomavirus (HPV) genotyping. Associations were examined between microbial prevalence, clinical characteristics, and p16 and EGFRmore » expression. Retroviral DNA was observed in 85% squamous cell carcinomas, 47% adenocarcinomas, and 10% adenocarcinomas with lepidic spread. Human papillomavirus DNA was found in 69% of squamous cell carcinomas with 30% containing high-risk HPV types. No significant viral DNA was detected in non-neoplastic lung. Patients with tumours containing viral DNA experienced improved long-term survival compared with patients with viral DNA-negative tumours. Lastly, most squamous cell carcinomas and adenocarcinomas contained retroviral DNA and one-third of squamous cell carcinomas contained high-risk HPV DNA. Viral DNA was absent in non-neoplastic lung. Trial results encourage further study of the viral contribution to lung carcinogenesis.« less

Molecular evidence of viral DNA in non-small cell lung cancer and non-neoplastic lung

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

Robinson, Lary A.; Jaing, Crystal J.; Campbell, Christine Pierce

Although ~20% of human cancers are caused by microorganisms, only suspicion exists for a microbial cause of lung cancer. Potential infectious agents were investigated in non-small cell lung cancer (NSCLC) and non-neoplastic lung. Seventy NSCLC tumours (33 squamous cell carcinomas, 17 adenocarcinomas, 10 adenocarcinomas with lepidic spread, and 10 oligometastases) and 10 non-neoplastic lung specimens were evaluated for molecular evidence of microorganisms. Tissues were subjected to the Lawrence Livermore Microbial Detection Array, an oncovirus panel of the International Agency for Research on Cancer, and human papillomavirus (HPV) genotyping. Associations were examined between microbial prevalence, clinical characteristics, and p16 and EGFRmore » expression. Retroviral DNA was observed in 85% squamous cell carcinomas, 47% adenocarcinomas, and 10% adenocarcinomas with lepidic spread. Human papillomavirus DNA was found in 69% of squamous cell carcinomas with 30% containing high-risk HPV types. No significant viral DNA was detected in non-neoplastic lung. Patients with tumours containing viral DNA experienced improved long-term survival compared with patients with viral DNA-negative tumours. Lastly, most squamous cell carcinomas and adenocarcinomas contained retroviral DNA and one-third of squamous cell carcinomas contained high-risk HPV DNA. Viral DNA was absent in non-neoplastic lung. Trial results encourage further study of the viral contribution to lung carcinogenesis.« less

Differences in Mouse and Human Non-Memory B Cell Pools1

PubMed Central

Benitez, Abigail; Weldon, Abby J.; Tatosyan, Lynnette; Velkuru, Vani; Lee, Steve; Milford, Terry-Ann; Francis, Olivia L.; Hsu, Sheri; Nazeri, Kavoos; Casiano, Carlos M.; Schneider, Rebekah; Gonzalez, Jennifer; Su, Rui-Jun; Baez, Ineavely; Colburn, Keith; Moldovan, Ioana; Payne, Kimberly J.

2014-01-01

Identifying cross-species similarities and differences in immune development and function is critical for maximizing the translational potential of animal models. Co-expression of CD21 and CD24 distinguishes transitional and mature B cell subsets in mice. Here, we validate these markers for identifying analogous subsets in humans and use them to compare the non-memory B cell pools in mice and humans, across tissues, during fetal/neonatal and adult life. Among human CD19+IgM+ B cells, the CD21/CD24 schema identifies distinct populations that correspond to T1 (transitional 1), T2 (transitional 2), FM (follicular mature), and MZ (marginal zone) subsets identified in mice. Markers specific to human B cell development validate the identity of MZ cells and the maturation status of human CD21/CD24 non-memory B cell subsets. A comparison of the non-memory B cell pools in bone marrow (BM), blood, and spleen in mice and humans shows that transitional B cells comprise a much smaller fraction in adult humans than mice. T1 cells are a major contributor to the non-memory B cell pool in mouse BM where their frequency is more than twice that in humans. Conversely, in spleen the T1:T2 ratio shows that T2 cells are proportionally ∼8 fold higher in humans than mouse. Despite the relatively small contribution of transitional B cells to the human non-memory pool, the number of naïve FM cells produced per transitional B cell is 3-6 fold higher across tissues than in mouse. These data suggest differing dynamics or mechanisms produce the non-memory B cell compartments in mice and humans. PMID:24719464

5-Bromodeoxyuridine induced differentiation of a human small cell lung cancer cell line is associated with alteration of gene expression.

PubMed

Chen, Yuan; Pacyna-Gengelbach, Manuela; Deutschmann, Nicole; Ye, Fei; Petersen, Iver

2007-02-16

Small cell lung cancer (SCLC) appears to arise from neuroendocrine cells with the potential to differentiate into a variety of lung epithelial cell lineages. In order to investigate molecular events underlying the cell type transition in SCLC, we treated a SCLC cell line H526 with a differentiation inducing agent 5-bromodeoxyuridine (BrdU). The treatment led to a dramatic conversion from suspension cells to adherent cells exhibiting an epithelioid phenotype, which remarkably reduced the ability of colony formation in soft agar and suppressed the tumor growth rate in nude mice. The phenotypic transition was consistent with upregulation of surfactant protein C (SFTPC), thyroid transcription factor 1 (TTF-1), Connexin 26 (Cx26), insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1), as well as homeobox genes LAGY, PITX1, and HOXB2. Our data suggest that BrdU induced cell differentiation could be linked to the development of a less aggressively phenotype in small cell lung cancer.

Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells.

PubMed

Singh, Brajesh K; Li, Ni; Mark, Anna C; Mateo, Mathieu; Cattaneo, Roberto; Sinn, Patrick L

2016-08-01

Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection, and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically, monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results obtained with free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small-pore (0.4-μm) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large-pore (3.0-μm) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large-pore membranes but not small-pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4-dependent manner. Thus, both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE. Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-to-cell contact rather than on particle release and reentry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-to-cell contact rather than through cell-free particles. In addition, we sought to determine which immune cells transfer MeV infectivity to the human airway epithelium. Our studies are based on two types of human primary cells: (i) myeloid cells generated from donated blood and (ii) well-differentiated airway epithelial cells derived from donor lungs. We show that different types of myeloid cells, i.e., monocyte-derived macrophages and dendritic cells, transfer infection to airway epithelial cells. Furthermore, cell-to-cell contact is an important component of successful MeV transfer. Our studies elucidate a mechanism by which the most contagious human respiratory virus is delivered to the airway epithelium. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Human parvovirus B19: a mechanistic overview of infection and DNA replication

PubMed Central

Luo, Yong; Qiu, Jianming

2015-01-01

Human parvovirus B19 (B19V) is a human pathogen that belongs to genus Erythroparvovirus of the Parvoviridae family, which is composed of a group of small DNA viruses with a linear single-stranded DNA genome. B19V mainly infects human erythroid progenitor cells and causes mild to severe hematological disorders in patients. However, recent clinical studies indicate that B19V also infects nonerythroid lineage cells, such as myocardial endothelial cells, and may be associated with other disease outcomes. Several cell culture systems, including permissive and semipermissive erythroid lineage cells, nonpermissive human embryonic kidney 293 cells and recently reported myocardial endothelial cells, have been used to study the mechanisms underlying B19V infection and B19V DNA replication. This review aims to summarize recent advances in B19V studies with a focus on the mechanisms of B19V tropism specific to different cell types and the cellular pathways involved in B19V DNA replication including cellular signaling transduction and cell cycle arrest. PMID:26097496

Enteroviruses infect human enteroids and induce antiviral signaling in a cell lineage-specific manner.

PubMed

Drummond, Coyne G; Bolock, Alexa M; Ma, Congrong; Luke, Cliff J; Good, Misty; Coyne, Carolyn B

2017-02-14

Enteroviruses are among the most common viral infectious agents of humans and are primarily transmitted by the fecal-oral route. However, the events associated with enterovirus infections of the human gastrointestinal tract remain largely unknown. Here, we used stem cell-derived enteroids from human small intestines to study enterovirus infections of the intestinal epithelium. We found that enteroids were susceptible to infection by diverse enteroviruses, including echovirus 11 (E11), coxsackievirus B (CVB), and enterovirus 71 (EV71), and that contrary to an immortalized intestinal cell line, enteroids induced antiviral and inflammatory signaling pathways in response to infection in a virus-specific manner. Furthermore, using the Notch inhibitor dibenzazepine (DBZ) to drive cellular differentiation into secretory cell lineages, we show that although goblet cells resist E11 infection, enteroendocrine cells are permissive, suggesting that enteroviruses infect specific cell populations in the human intestine. Taken together, our studies provide insights into enterovirus infections of the human intestine, which could lead to the identification of novel therapeutic targets and/or strategies to prevent or treat infections by these highly clinically relevant viruses.

TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells

NASA Astrophysics Data System (ADS)

De Miguel, Diego; Gallego-Lleyda, Ana; María Ayuso, José; Erviti-Ardanaz, Sandra; Pazo-Cid, Roberto; del Agua, Celia; José Fernández, Luis; Ochoa, Ignacio; Anel, Alberto; Martinez-Lostao, Luis

2016-05-01

Purpose. Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. Methods/patients. LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. Results. LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. Conclusion. The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment.

TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells.

PubMed

De Miguel, Diego; Gallego-Lleyda, Ana; Ayuso, José María; Erviti-Ardanaz, Sandra; Pazo-Cid, Roberto; del Agua, Celia; Fernández, Luis José; Ochoa, Ignacio; Anel, Alberto; Martinez-Lostao, Luis

2016-05-06

Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment.

Expression of GFP under the control of the RNA helicase VASA permits fluorescence-activated cell sorting isolation of human primordial germ cells.

PubMed

Tilgner, Katarzyna; Atkinson, Stuart P; Yung, Sun; Golebiewska, Anna; Stojkovic, Miodrag; Moreno, Ruben; Lako, Majlinda; Armstrong, Lyle

2010-01-01

The isolation of significant numbers of human primordial germ cells at several developmental stages is important for investigations of the mechanisms by which they are able to undergo epigenetic reprogramming. Only small numbers of these cells can be obtained from embryos of appropriate developmental stages, so the differentiation of human embryonic stem cells is essential to obtain sufficient numbers of primordial germ cells to permit epigenetic examination. Despite progress in the enrichment of human primordial germ cells using fluorescence-activated cell sorting (FACS), there is still no definitive marker of the germ cell phenotype. Expression of the widely conserved RNA helicase VASA is restricted to germline cells, but in contrast to species such as Mus musculus in which reporter constructs expressing green fluorescent protein (GFP) under the control of a Vasa promoter have been developed, such reporter systems are lacking in human in vitro models. We report here the generation and characterization of human embryonic stem cell lines stably carrying a VASA-pEGFP-1 reporter construct that expresses GFP in a population of differentiating human embryonic stem cells that show expression of characteristic markers of primordial germ cells. This population shows a different pattern of chromatin modifications to those obtained by FACS enrichment of Stage Specific Antigen one expressing cells in our previous publication.

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation.

PubMed

Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

2015-12-01

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

PubMed Central

Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

2016-01-01

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies. PMID:26587712

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

NASA Astrophysics Data System (ADS)

Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

2015-12-01

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Role of Cell-to-Cell Variability in Activating a Positive Feedback Antiviral Response in Human Dendritic Cells

PubMed Central

Hu, Jianzhong; Nudelman, German; Shimoni, Yishai; Kumar, Madhu; Ding, Yaomei; López, Carolina; Hayot, Fernand; Wetmur, James G.; Sealfon, Stuart C.

2011-01-01

In the first few hours following Newcastle disease viral infection of human monocyte-derived dendritic cells, the induction of IFNB1 is extremely low and the secreted type I interferon response is below the limits of ELISA assay. However, many interferon-induced genes are activated at this time, for example DDX58 (RIGI), which in response to viral RNA induces IFNB1. We investigated whether the early induction of IFNBI in only a small percentage of infected cells leads to low level IFN secretion that then induces IFN-responsive genes in all cells. We developed an agent-based mathematical model to explore the IFNBI and DDX58 temporal dynamics. Simulations showed that a small number of early responder cells provide a mechanism for efficient and controlled activation of the DDX58-IFNBI positive feedback loop. The model predicted distributions of single cell responses that were confirmed by single cell mRNA measurements. The results suggest that large cell-to-cell variation plays an important role in the early innate immune response, and that the variability is essential for the efficient activation of the IFNB1 based feedback loop. PMID:21347441

Variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates.

PubMed

Werner, Benjamin; Sottoriva, Andrea

2018-06-01

The immortal strand hypothesis poses that stem cells could produce differentiated progeny while conserving the original template strand, thus avoiding accumulating somatic mutations. However, quantitating the extent of non-random DNA strand segregation in human stem cells remains difficult in vivo. Here we show that the change of the mean and variance of the mutational burden with age in healthy human tissues allows estimating strand segregation probabilities and somatic mutation rates. We analysed deep sequencing data from healthy human colon, small intestine, liver, skin and brain. We found highly effective non-random DNA strand segregation in all adult tissues (mean strand segregation probability: 0.98, standard error bounds (0.97,0.99)). In contrast, non-random strand segregation efficiency is reduced to 0.87 (0.78,0.88) in neural tissue during early development, suggesting stem cell pool expansions due to symmetric self-renewal. Healthy somatic mutation rates differed across tissue types, ranging from 3.5 × 10-9/bp/division in small intestine to 1.6 × 10-7/bp/division in skin.

Cryopreservation of Human Pluripotent Stem Cells in Defined Medium

PubMed Central

Liu, Weiwei; Chen, Guokai

2014-01-01

This protocol describes a cryopreservation procedure using an enzyme-free dissociation method to harvest cells and preserve cells in albumin-free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. The preservation in E8 medium eliminates serum or other animal products, and is suitable for the increasing demand for high quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, this protocol allows efficient cryopreservation in more time-saving manner. PMID:25366897

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Meerovich, Irina G.; Arslanbaeva, Lyaisan R.; Jerdeva, Viktoria V.; Orlova, Anna G.; Kleshnin, Mikhail S.; Shirmanova, Marina V.; Fiks, Ilya I.

2007-02-01

Conventional optical imaging is restricted with tumor size due to high tissue scattering. Labeling of tumors by fluorescent markers improves sensitivity of tumor detection thus increasing the value of optical imaging dramatically. Creation of tumor cell lines transfected with fluorescent proteins gives the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and TurboRFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

Fluorescence diffuse tomography for detection of RFP-expressed tumors in small animals

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Orlova, Anna G.; Kleshnin, Mikhail S.; Shirmanova, Marina V.; Fix, Ilya I.; Popov, Vladimir O.

2007-07-01

Capabilities of tumor detection by different optical methods can be significantly improved by labeling of tumors with fluorescent markers. Creation of tumor cell lines transfected with fluorescent proteins provides the possibility not only to detect tumor, but also to conduct the intravital monitoring studies. Cell lines of human melanomas Mel-P, Mel-Kor and human embryonic kidney HEK-293 Phoenix were transfected with DsRed-Express and Turbo-RFP genes. Emission of RFP in the long-wave optical range permits detection of the deeply located tumors, which is essential for whole-body imaging. Only special tools for turbid media imaging, such as fluorescent diffusion tomography (FDT), enable noninvasive investigation of the internal structure of biological tissue. FDT setup for monitoring of tumor growth in small animals has been created. An animal is scanned in the transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at the 532 nm wavelength. An optimizing algorithm for scanning of an experimantal animal is suggested. In vivo experiments were conducted immediately after the subcutaneously injection of fluorescing cells into small animals. It was shown that FDT method allows to detect the presence of fluorescent cells in small animals and can be used for monitoring of tumor growth and anticancer drug responce.

Combining cell sheet technology and electrospun scaffolding for engineered tubular, aligned, and contractile blood vessels.

PubMed

Rayatpisheh, Shahrzad; Heath, Daniel E; Shakouri, Amir; Rujitanaroj, Pim-On; Chew, Sing Yian; Chan-Park, Mary B

2014-03-01

Herein we combine cell sheet technology and electrospun scaffolding to rapidly generate circumferentially aligned tubular constructs of human aortic smooth muscles cells with contractile gene expression for use as tissue engineered blood vessel media. Smooth muscle cells cultured on micropatterned and N-isopropylacrylamide-grafted (pNIPAm) polydimethylsiloxane (PDMS), a small portion of which was covered by aligned electrospun scaffolding, resulted in a single sheet of unidirectionally aligned cells. Upon cooling to room temperature, the scaffold, its adherent cells, and the remaining cell sheet detached and were collected on a mandrel to generating tubular constructs with circumferentially aligned smooth muscle cells which possess contractile gene expression and a single layer of electrospun scaffold as an analogue to a small diameter blood vessel's internal elastic lamina (IEL). This method improves cell sheet handling, results in rapid circumferential alignment of smooth muscle cells which immediately express contractile genes, and introduction of an analogue to small diameter blood vessel IEL. Copyright © 2013 Elsevier Ltd. All rights reserved.

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine

PubMed Central

Muglia, C; Mercer, N; Toscano, M A; Schattner, M; Pozner, R; Cerliani, J P; Gobbi, R Papa; Rabinovich, G A; Docena, G H

2011-01-01

Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals1−/−) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals1−/− mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function. PMID:21614093

Expression and function of Allergin-1 on human primary mast cells.

PubMed

Nagai, Kei; Tahara-Hanaoka, Satoko; Morishima, Yuko; Tokunaga, Takahiro; Imoto, Yoshimasa; Noguchi, Emiko; Kanemaru, Kazumasa; Imai, Masamichi; Shibayama, Shiro; Hizawa, Nobuyuki; Fujieda, Shigeharu; Yamagata, Kunihiro; Shibuya, Akira

2013-01-01

Mast cells (MC) play an important role in allergic and non-allergic immune responses. Activation of human MC is modulated by several cell surface inhibitory receptors, including recently identified Allergin-1 expressed on both human and mouse MC. Although Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice, the expression profile and function of Allergin-1 on human primary MC remains undetermined. Here, we established a seven-color flow cytometry method for assessing expression and function of a very small number of human primary MC. We show that Allergin-1S1, a splicing isoform of Allergin-1, is predominantly expressed on human primary MC in both bronchoalveolar lavage (BAL) fluid and nasal scratching specimens. Moreover, Allergin-1S1 inhibits IgE-mediated activation from human primary MC in BAL fluid. These results indicate that Allergin-1 on human primary MC exhibits similar characteristics as mouse Allergin-1 in the expression profile and function.

FLT-PET/CT as a Biomarker of Therapeutic Response in Pemetrexed Therapy for Non-Small Cell Lung Cancer

DTIC Science & Technology

2015-10-01

therapy in a human patient. These images are from a 63 y/o male with NSCLC participating in our exploratory clinical trial of FLT- PET “flare”. (a... PET in a human NSCLC patient. Results: In NSCLC cells in vitro, we identified a burst in thymidine salvage pathway activity, assessed by 3H...25 Human PET imaging IRB approval was obtained through the University of Pennsylvania Institutional Review Board for use of FLT- PET /CT

Immunohistopathology of Prototheca wickerhamii in cutaneous lesions of protothecosis.

PubMed

Kano, Rui; Sobukawa, Hideto; Suzuki, Minako; Hiruma, Masataro; Shibuya, Kazutoshi; Hasegawa, Atsuhiko; Kamata, Hiroshi

2014-01-01

Protothecosis is a rare infection caused by pathogenic algae of the genus Prototheca. Prototheca wickerhamii causes cutaneous/subcutaneous opportunistic infections in humans and small animals. The diagnosis of protothecosis is based on histopathological examination of this organism, which can be confused with other fungi and inflammatory cells in infected tissues. In this study, immunohistopathological investigation was made of infected cutaneous human and animal tissues exhibiting protothecosis using rabbit antiserum against P. wickerhamii. Serum detected P. wickerhamii in human and feline protothecosis tissues, and did not react with Candida albicans in the human kidney tissues showing candidiasis. This antiserum can therefore differentiate P. wickerhamii cells from the yeast-like cells of C. albicans and Prototheca zopfii in target tissues.

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 beta-dystroglycan is reduced or absent in many human carcinomas.

PubMed

Cross, S S; Lippitt, J; Mitchell, A; Hollingsbury, F; Balasubramanian, S P; Reed, M W R; Eaton, C; Catto, J W; Hamdy, F; Winder, S J

2008-11-01

Dystroglycan is an important structural and signalling protein that is expressed in most human cells. alpha-Dystroglycan has been investigated and found to be reduced in human cancers, but there is only one published study on the expression of beta-dystroglycan in human cancer and that was only on small numbers of breast and prostatic cancers. The aim was to conduct a comprehensive immunohistochemical survey of the expression of beta-dystroglycan in normal human tissues and common cancers. Triplicate tissue microarrays of 681 samples of normal human tissues and common cancers were stained using an antibody directed against the cytoplasmic component of beta-dystroglycan. beta-Dystroglycan was strongly expressed at the intercellular junctions and basement membranes of all normal human epithelia. Expression of beta-dystroglycan was absent or markedly reduced in 100% of oesophageal adenocarcinomas, 97% of colonic cancers, 100% of transitional cell carcinomas of the urothelium and 94% of breast cancers. In the breast cancers, the only tumours that showed any retention of beta-dystroglycan expression were small low-grade oestrogen receptor-positive tumours. The only cancers that showed retention of beta-dystroglycan expression were cutaneous basal cell carcinomas. There is loss or marked reduction of beta-dystroglycan expression (by immunohistochemistry) in the vast majority of human cancers surveyed. Since beta-dystroglycan is postulated to have a tumour suppressor effect, this loss may have important functional significance.

Glycogen Synthase Kinase 3 Protein Kinase Activity Is Frequently Elevated in Human Non-Small Cell Lung Carcinoma and Supports Tumour Cell Proliferation

PubMed Central

O′Flaherty, Linda; Pardo, Olivier E.; Dzien, Piotr; Phillips, Lois; Morgan, Carys; Pawade, Joya; May, Margaret T.; Sohail, Muhammad; Hetzel, Martin R.; Seckl, Michael J.; Tavaré, Jeremy M.

2014-01-01

Background Glycogen synthase kinase 3 (GSK3) is a central regulator of cellular metabolism, development and growth. GSK3 activity was thought to oppose tumourigenesis, yet recent studies indicate that it may support tumour growth in some cancer types including in non-small cell lung carcinoma (NSCLC). We examined the undefined role of GSK3 protein kinase activity in tissue from human NSCLC. Methods The expression and protein kinase activity of GSK3 was determined in 29 fresh frozen samples of human NSCLC and patient-matched normal lung tissue by quantitative immunoassay and western blotting for the phosphorylation of three distinct GSK3 substrates in situ (glycogen synthase, RelA and CRMP-2). The proliferation and sensitivity to the small-molecule GSK3 inhibitor; CHIR99021, of NSCLC cell lines (Hcc193, H1975, PC9 and A549) and non-neoplastic type II pneumocytes was further assessed in adherent culture. Results Expression and protein kinase activity of GSK3 was elevated in 41% of human NSCLC samples when compared to patient-matched control tissue. Phosphorylation of GSK3α/β at the inhibitory S21/9 residue was a poor biomarker for activity in tumour samples. The GSK3 inhibitor, CHIR99021 dose-dependently reduced the proliferation of three NSCLC cell lines yet was ineffective against type II pneumocytes. Conclusion NSCLC tumours with elevated GSK3 protein kinase activity may have evolved dependence on the kinase for sustained growth. Our results provide further important rationale for exploring the use of GSK3 inhibitors in treating NSCLC. PMID:25486534

Developing Novel Therapeutic Approaches in Small Cell Lung Carcinoma Using Genetically Engineered Mouse Models and Human Circulating Tumor Cells

DTIC Science & Technology

2015-10-01

xenograft models . 12-36 Dr. Engelman Subtask 3: Analyze CTCs for P-4EBP1, P-S6, BIM , Bcl-2, Bcl-xL, and Mcl-1 using ISH and IHC We propose...Using Genetically Engineered Mouse Models and Human Circulating Tumor Cells PRINCIPAL INVESTIGATOR: Jeffrey Engelman MD PhD CONTRACTING...reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions

Brain microvascular endothelium induced-annexin A1 secretion contributes to small cell lung cancer brain metastasis.

PubMed

Liu, Yi; Liu, Yong-Shuo; Wu, Peng-Fei; Li, Qiang; Dai, Wu-Min; Yuan, Shuai; Xu, Zhi-Hua; Liu, Ting-Ting; Miao, Zi-Wei; Fang, Wen-Gang; Chen, Yu-Hua; Li, Bo

2015-09-01

Small cell lung cancer is the most aggressive histologic subtype of lung cancer, with a strong predilection for metastasizing to brain early. However, the cellular and molecular basis is poorly known. Here, we provided evidence to reveal the role of annexin A1 in small cell lung cancer metastasis to brain. Firstly, the elevated annexin A1 serum levels in small cell lung cancer patients were associated with brain metastasis. The levels of annexin A1 were also upregulated in NCI-H446 cells, a small cell lung cancer cell line, upon migration into the mice brain. More interestingly, annexin A1 was secreted by NCI-H446 cells in a time-dependent manner when co-culturing with human brain microvascular endothelial cells, which was identified with the detections of annexin A1 in the co-cultured cellular supernatants by ELISA and western blot. Further results showed that blockage of annexin A1 in the co-cultured cellular supernatants using a neutralized antibody significantly inhibited NCI-H446 cells adhesion to brain endothelium and its transendothelial migration. Conversely, the addition of Ac2-26, an annexin A1 mimic peptide, enhanced these effects. Furthermore, knockdown of annexin A1 in NCI-H446 cells prevented its transendothelial migration in vitro and metastasis to mice brain in vivo. Our data showed that small cell lung cancer cell in brain microvasculature microenvironment could express much more annexin A1 and release it outside, which facilitated small cell lung cancer cell to gain malignant properties of entry into brain. These findings provided a potential target for the management of SCLC brain metastasis. Copyright © 2015 Elsevier Ltd. All rights reserved.

A novel imidazopyridine PI3K inhibitor with anticancer activity in non-small cell lung cancer cells.

PubMed

Lee, Hyunseung; Kim, Soo Jung; Jung, Kyung Hee; Son, Mi Kwon; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

2013-08-01

Lung cancer is the leading cause of cancer-related mortality in the world, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases. Since more than 60% of NSCLC cases express the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors are used to treat NSCLC. However, due to the acquired resistance associated with EGFR-targeted therapy, other strategies for the treatment of NSCLC are urgently needed. Therefore, we investigated the anticancer effects of a novel phosphatidylinositol 3-kinase α (PI3Kα) inhibitor, HS-173, in human NSCLC cell lines. HS-173 demonstrated anti-proliferative effects in NSCLC cells and effectively inhibited the PI3K signaling pathway in a dose‑dependent manner. In addition, it induced cell cycle arrest at G2/M phase as well as apoptosis. Taken together, our results demonstrate that HS-173 exhibits anticancer activities, including the induction of apoptosis, by blocking the PI3K/Akt/mTOR pathway in human NSCLC cell lines. We, therefore, suggest that this novel drug could potentially be used for targeted NSCLC therapy.

A small molecule-based strategy for endothelial differentiation and three-dimensional morphogenesis from human embryonic stem cells.

PubMed

Geng, Yijie; Feng, Bradley

2016-07-01

The emerging models of human embryonic stem cell (hESC) self-organizing organoids provide a valuable in vitro platform for studying self-organizing processes that presumably mimic in vivo human developmental events. Here we report that through a chemical screen, we identified two novel and structurally similar small molecules BIR1 and BIR2 which robustly induced the self-organization of a balloon-shaped three-dimensional structure when applied to two-dimensional adherent hESC cultures in the absence of growth factors. Gene expression analyses and functional assays demonstrated an endothelial identity of this balloon-like structure, while cell surface marker analyses revealed a VE-cadherin(+)CD31(+)CD34(+)KDR(+)CD43(-) putative endothelial progenitor population. Furthermore, molecular marker labeling and morphological examinations characterized several other distinct DiI-Ac-LDL(+) multi-cellular modules and a VEGFR3(+) sprouting structure in the balloon cultures that likely represented intermediate structures of balloon-formation.

Protein Kinase-A Inhibition Is Sufficient to Support Human Neural Stem Cells Self-Renewal.

PubMed

Georges, Pauline; Boissart, Claire; Poulet, Aurélie; Peschanski, Marc; Benchoua, Alexandra

2015-12-01

Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro. © 2015 AlphaMed Press.

Involvement of aryl hydrocarbon receptor signaling in the development of small cell lung cancer induced by HPV E6/E7 oncoproteins

PubMed Central

2011-01-01

Background Lung cancers consist of four major types that and for clinical-pathological reasons are often divided into two broad categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). All major histological types of lung cancer are associated with smoking, although the association is stronger for SCLC and squamous cell carcinoma than adenocarcinoma. To date, epidemiological studies have identified several environmental, genetic, hormonal and viral factors associated with lung cancer risk. It has been estimated that 15-25% of human cancers may have a viral etiology. The human papillomavirus (HPV) is a proven cause of most human cervical cancers, and might have a role in other malignancies including vulva, skin, oesophagus, head and neck cancer. HPV has also been speculated to have a role in the pathogenesis of lung cancer. To validate the hypothesis of HPV involvement in small cell lung cancer pathogenesis we performed a gene expression profile of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins. Methods Gene expression profile of SCLC has been performed using Agilent whole mouse genome (4 × 44k) representing ~ 41000 genes and mouse transcripts. Samples were obtained from two HPV16-E6/E7 transgenic mouse models and from littermate's normal lung. Data analyses were performed using GeneSpring 10 and the functional classification of deregulated genes was performed using Ingenuity Pathway Analysis (Ingenuity® Systems, http://www.ingenuity.com). Results Analysis of deregulated genes induced by the expression of E6/E7 oncoproteins supports the hypothesis of a linkage between HPV infection and SCLC development. As a matter of fact, comparison of deregulated genes in our system and those in human SCLC showed that many of them are located in the Aryl Hydrocarbon Receptor Signal transduction pathway. Conclusions In this study, the global gene expression of transgenic mouse model of SCLC induced by HPV-16 E6/E7 oncoproteins led us to identification of several genes involved in SCLC tumor development. Furthermore, our study reveled that the Aryl Hydrocarbon Receptor Signaling is the primarily affected pathway by the E6/E7 oncoproteins expression and that this pathway is also deregulated in human SCLC. Our results provide the basis for the development of new therapeutic approaches against human SCLC. PMID:21205295

A Mathematical Model of the Human Small Intestine Following Acute Radiation and Burn Exposures

DTIC Science & Technology

2016-08-01

Acronyms and Symbols ARA Applied Research Associates, Inc. ARS Acute radiation syndrome d Days DE Differential Evolution DTRA Defense Threat...04-08-2016 Technical Report A Mathematical Model of the Human Small Intestine Following Acute Radiation and Burn Exposures HDTRA1...epithelial cells to acute radiation alone. The model has been modified for improved radiation response, and an addition to the model allows for thermal injury

TUSC3 induces autophagy in human non-small cell lung cancer cells through Wnt/β-catenin signaling

PubMed Central

Peng, Yun; Cao, Jun; Yao, Xiao-Yi; Wang, Jian-Xin; Zhong, Mei-Zuo; Gan, Ping-Ping; Li, Jian-Huang

2017-01-01

We investigated the effects of tumor suppressor candidate 3 (TUSC3) on autophagy in human non-small cell lung cancer (NSCLC) cells. A total of 118 NSCLC patients (88 males and 30 females) who underwent surgery at our institute were enrolled in the study. Immunohistochemical analysis revealed that TUSC3 protein expression was lower in NSCLC specimens than adjacent normal tissue. Correspondingly, there was greater methylation of TUSC3 in NSCLC than adjacent normal tissue. After transient transfection of A549 NSCLC cells with constructs designed to up-regulate or down-regulate TUSC3 expression, we analyzed the effects of inhibiting the Wnt pathway (XAV939) and autophagy (chloroquine, CQ) on the behavior of NSCLC cells. We also performed TOP/FOP-Flash reporter assays, MTT assays, Annexin V-FITC/propidium iodide staining, and acridine orange staining to evaluate Wnt/β-catenin signaling, cell proliferation, apoptosis, and autophagy, respectively. Expression of Wnt/β-catenin pathway components and autophagy-related proteins was analyzed using qRT-PCR and Western blotting. We found that TUSC3 inhibited cell proliferation and promoted both apoptosis and autophagy in A549 cells. In addition, TUSC3 increased expression of autophagy-related proteins. It also increased expression of Wnt/β-catenin signaling pathway components and promoted nuclear transfer of β-catenin, resulting in activation of Wnt/β-catenin signaling. TUSC3 thus induces autophagy in human NSCLC cells through activation of the Wnt/β-catenin signaling pathway. PMID:28881786

TUSC3 induces autophagy in human non-small cell lung cancer cells through Wnt/β-catenin signaling.

PubMed

Peng, Yun; Cao, Jun; Yao, Xiao-Yi; Wang, Jian-Xin; Zhong, Mei-Zuo; Gan, Ping-Ping; Li, Jian-Huang

2017-08-08

We investigated the effects of tumor suppressor candidate 3 ( TUSC3 ) on autophagy in human non-small cell lung cancer (NSCLC) cells. A total of 118 NSCLC patients (88 males and 30 females) who underwent surgery at our institute were enrolled in the study. Immunohistochemical analysis revealed that TUSC3 protein expression was lower in NSCLC specimens than adjacent normal tissue. Correspondingly, there was greater methylation of TUSC3 in NSCLC than adjacent normal tissue. After transient transfection of A549 NSCLC cells with constructs designed to up-regulate or down-regulate TUSC3 expression, we analyzed the effects of inhibiting the Wnt pathway (XAV939) and autophagy (chloroquine, CQ) on the behavior of NSCLC cells. We also performed TOP/FOP-Flash reporter assays, MTT assays, Annexin V-FITC/propidium iodide staining, and acridine orange staining to evaluate Wnt/β-catenin signaling, cell proliferation, apoptosis, and autophagy, respectively. Expression of Wnt/β-catenin pathway components and autophagy-related proteins was analyzed using qRT-PCR and Western blotting. We found that TUSC3 inhibited cell proliferation and promoted both apoptosis and autophagy in A549 cells. In addition, TUSC3 increased expression of autophagy-related proteins. It also increased expression of Wnt/β-catenin signaling pathway components and promoted nuclear transfer of β-catenin, resulting in activation of Wnt/β-catenin signaling. TUSC3 thus induces autophagy in human NSCLC cells through activation of the Wnt/β-catenin signaling pathway.

Transcriptional Inhibition of the Human Papilloma Virus Reactivates Tumor Suppressor p53 in Cervical Carcinoma Cells

PubMed Central

Kochetkov, D. V.; Ilyinskaya, G. V.; Komarov, P. G.; Strom, E.; Agapova, L. S.; Ivanov, A. V.; Budanov, A. V.; Frolova, E. I.; Chumakov, P. M.

2009-01-01

Inactivation of tumor suppressor p53 accompanies the majority of human malignancies. Restoration of p53 function causes death of tumor cells and is potentially suitable for gene therapy of cancer. In cervical carcinoma, human papilloma virus (HPV) E6 facilitates proteasomal degradation of p53. Hence, a possible approach to p53 reactivation is the use of small molecules suppressing the function of viral proteins. HeLa cervical carcinoma cells (HPV-18) with a reporter construct containing the b-galactosidase gene under the control of a p53-responsive promoter were used as a test system to screen a library of small molecules for restoration of the transcriptional activity of p53. The effect of the two most active compounds was studied with cell lines differing in the state of p53-dependent signaling pathways. The compounds each specifically activated p53 in cells expressing HPV-18 and, to a lesser extent, HPV-16 and exerted no effect on control p53-negative cells or cells with the intact p53-dependent pathways. Activation of p53 in cervical carcinoma cells was accompanied by induction of p53-dependent CDKN1 (p21), inhibition of cell proliferation, and induction of apoptosis. In addition, the two compounds dramatically decreased transcription of the HPV genome, which was assumed to cause p53 reactivation. The compounds were low-toxic for normal cells and can be considered as prototypes of new anticancer drugs. PMID:17685229

An inducible caspase 9 safety switch for T-cell therapy

PubMed Central

Straathof, Karin C.; Pulè, Martin A.; Yotnda, Patricia; Dotti, Gianpietro; Vanin, Elio F.; Brenner, Malcolm K.; Heslop, Helen E.; Spencer, David M.; Rooney, Cliona M.

2005-01-01

The efficacy of adoptive T-cell therapy as treatment for malignancies may be enhanced by genetic modification of infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves, have tempered enthusiasm. A safe and efficient means of removing aberrant cells in vivo would ameliorate these concerns. We describe a “safety switch” that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This reagent is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using a small molecule pharmaceutical. A single 10-nM dose of synthetic dimerizer drug induces apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo. This system has several advantages over currently available suicide genes. First, it consists of human gene products with low potential immunogenicity. Second, administration of dimerizer drug has no effects other than the selective elimination of transduced T cells. Third, inducible caspase 9 maintains function in T cells overexpressing antiapoptotic molecules. These characteristics favor incorporation of inducible caspase 9 as a safety feature in human T-cell therapies. PMID:15728125

Effects of Laser Printer–Emitted Engineered Nanoparticles on Cytotoxicity, Chemokine Expression, Reactive Oxygen Species, DNA Methylation, and DNA Damage: A Comprehensive in Vitro Analysis in Human Small Airway Epithelial Cells, Macrophages, and Lymphoblasts

PubMed Central

Pirela, Sandra V.; Miousse, Isabelle R.; Lu, Xiaoyan; Castranova, Vincent; Thomas, Treye; Qian, Yong; Bello, Dhimiter; Kobzik, Lester; Koturbash, Igor; Demokritou, Philip

2015-01-01

Background Engineered nanomaterials (ENMs) incorporated into toner formulations of printing equipment become airborne during consumer use. Although information on the complex physicochemical and toxicological properties of both toner powders and printer-emitted particles (PEPs) continues to grow, most toxicological studies have not used the actual PEPs but rather have primarily used raw toner powders, which are not representative of current exposures experienced at the consumer level during printing. Objectives We assessed the biological responses of a panel of human cell lines to PEPs. Methods Three physiologically relevant cell lines—small airway epithelial cells (SAECs), macrophages (THP-1 cells), and lymphoblasts (TK6 cells)—were exposed to PEPs at a wide range of doses (0.5–100 μg/mL) corresponding to human inhalation exposure durations at the consumer level of 8 hr or more. Following treatment, toxicological parameters reflecting distinct mechanisms were evaluated. Results PEPs caused significant membrane integrity damage, an increase in reactive oxygen species (ROS) production, and an increase in pro-inflammatory cytokine release in different cell lines at doses equivalent to exposure durations from 7.8 to 1,500 hr. Furthermore, there were differences in methylation patterns that, although not statistically significant, demonstrate the potential effects of PEPs on the overall epigenome following exposure. Conclusions The in vitro findings obtained in this study suggest that laser printer–emitted engineered nanoparticles may be deleterious to lung cells and provide preliminary evidence of epigenetic modifications that might translate to pulmonary disorders. Citation Pirela SV, Miousse IR, Lu X, Castranova V, Thomas T, Qian Y, Bello D, Kobzik L, Koturbash I, Demokritou P. 2016. Effects of laser printer–emitted engineered nanoparticles on cytotoxicity, chemokine expression, reactive oxygen species, DNA methylation, and DNA damage: a comprehensive in vitro analysis in human small airway epithelial cells, macrophages, and lymphoblasts. Environ Health Perspect 124:210–219; http://dx.doi.org/10.1289/ehp.1409582 PMID:26080392

The 57Fe hyperfine interactions in iron storage proteins in liver and spleen tissues from normal human and two patients with mantle cell lymphoma and acute myeloid leukemia: a Mössbauer effect study

NASA Astrophysics Data System (ADS)

Oshtrakh, M. I.; Alenkina, I. V.; Vinogradov, A. V.; Konstantinova, T. S.; Semionkin, V. A.

2015-04-01

Study of human spleen and liver tissues from healthy persons and two patients with mantle cell lymphoma and acute myeloid leukemia was carried out using Mössbauer spectroscopy with a high velocity resolution. Small variations in the 57Fe hyperfine parameters for normal and patient's tissues were detected and related to small variations in the 57Fe local microenvironment in ferrihydrite cores. The differences in the relative parts of more crystalline and more amorphous core regions were also supposed for iron storage proteins in normal and patients' spleen and liver tissues.

Assessment of oral transmission using cell-free human immunodeficiency virus-1 in mice reconstituted with human peripheral blood leucocyte

PubMed Central

Nakao, Ryoma; Hanada, Nobuhiro; Asano, Toshihiko; Hara, Takashi; Abdus Salam, MD; Matin, Khairul; Shimazu, Yoshihito; Nakasone, Tadashi; Horibata, Shigeo; Aoba, Takaaki; Honda, Mitsuo; Amagasa, Teruo; Senpuku, Hidenobu

2003-01-01

Oral–genital contact is one of the risk factors for the transmission of human immunodeficiency virus (HIV) in adults. In recent reports, oral exposure to simian immunodeficiency virus (SIV) was found to have important implications for the achievement of mucosal transmission of HIV in a rhesus monkey animal model. In the present study, we aimed first to establish a small animal model which did not develop tonsils suitable for HIV oral mucosa transmission, using non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and NOD/SCID B2mnull mice grafted with human peripheral blood leucocytes (hu-PBL) and stimulated with interleukin (IL)-4, and second to investigate whether oral exposure to cell-free R5 and X4 HIV-1 could lead to oral transmission of HIV through intact or traumatized mucosal tissues in humanized mice. Both low and high concentrations of cell-free R5 and X4 viruses failed to cause oral transmission with or without trauma in hu-PBL-NOD/SCID and NOD/SCID Β2mnull mice, which presented a number of CD4+ cells in gingival tissues and oral cavities with or without tissue injury. The present results show that IL-4-administrated NOD/SCID B2mnull mice are useful as a small-humanized model for the study of HIV oral infection, which may help to define the window of opportunity for oral transmission by the HIV virus in animal model experiments. PMID:12757623

The anticancer natural product ophiobolin A induces cytotoxicity by covalent modification of phosphatidylethanolamine.

PubMed

Chidley, Christopher; Trauger, Sunia A; Birsoy, Kıvanç; O'Shea, Erin K

2016-07-12

Phenotypic screens allow the identification of small molecules with promising anticancer activity, but the difficulty in characterizing the mechanism of action of these compounds in human cells often undermines their value as drug leads. Here, we used a loss-of-function genetic screen in human haploid KBM7 cells to discover the mechanism of action of the anticancer natural product ophiobolin A (OPA). We found that genetic inactivation of de novo synthesis of phosphatidylethanolamine (PE) mitigates OPA cytotoxicity by reducing cellular PE levels. OPA reacts with the ethanolamine head group of PE in human cells to form pyrrole-containing covalent cytotoxic adducts and these adducts lead to lipid bilayer destabilization. Our characterization of this unusual cytotoxicity mechanism, made possible by unbiased genetic screening in human cells, suggests that the selective antitumor activity displayed by OPA may be due to altered membrane PE levels in cancer cells.

Recent progress in human telomere RNA structure and function.

PubMed

Xu, Yan

2018-06-14

Human telomeric DNA is transcribed into telomeric RNA in cells. Telomeric RNA performs the fundamental biological functions such as regulation and protection of chromosome ends. This digest highlights the human telomere RNA G-quadruplex structures, telomere RNA functions, G-quadruplex-binding small molecules, and future prospects. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neurotensin receptors in human neoplasms: high incidence in Ewing's sarcomas.

PubMed

Reubi, J C; Waser, B; Schaer, J C; Laissue, J A

1999-07-19

Receptors for regulatory peptides, such as somatostatin or vasoactive intestinal peptide (VIP), expressed at high density by neoplastic cells, can be instrumental for tumor diagnosis and therapy. Little is known about the expression of neurotensin receptors in human tumors. In the present study, 464 human neoplasms of various types were investigated for their neurotensin receptor content by in vitro receptor autoradiography on tissue sections using 125I-[Tyr3]-neurotensin as radioligand. Neurotensin receptors were identified and localized in tumor cells of 11/17 Ewing's sarcomas, 21/40 meningiomas, 10/23 astrocytomas, 5/13 medulloblastomas, 7/24 medullary thyroid cancers and 2/8 small cell lung cancers. They were rarely found in non-small cell lung cancers and breast carcinomas; they were absent in prostate, ovarian, renal cell and hepatocellular carcinomas, neuroendocrine gut tumors, pituitary adenomas, schwannomas, neuroblastomas and lymphomas. When present, the receptors bound with nanomolar affinity neurotensin and acetyl-neurotensin-(8-13), with lower affinity neuromedin N, diethylenetriamine penta-acetic acidneurotensin-(8-13) and SR 48692, but not neurotensin-(1-11). They were all of the NT1 type, without high affinity for levocabastine. Further, in 2 receptor-positive Ewing's sarcomas, neurotensin mRNA was detected by in situ hybridization techniques. Since neurotensin is known to stimulate cell proliferation, the presence of neurotensin receptors in human neoplasia may be of biological relevance, possibly as an integrative part of an autocrine feedback mechanism of tumor growth stimulation.

Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage.

PubMed Central

Warren, S T; Knight, S J; Peters, J F; Stayton, C L; Consalez, G G; Zhang, F P

1990-01-01

The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region. Images PMID:2339126

Identification of a coumarin-based antihistamine-like small molecule as an anti-filoviral entry inhibitor.

PubMed

Cheng, Han; Schafer, Adam; Soloveva, Veronica; Gharaibeh, Dima; Kenny, Tara; Retterer, Cary; Zamani, Rouzbeh; Bavari, Sina; Peet, Norton P; Rong, Lijun

2017-09-01

Filoviruses, consisting of Ebola virus, Marburg virus and Cuevavirus, cause severe hemorrhagic fevers in humans with high mortality rates up to 90%. Currently, there is no approved vaccine or therapy available for the prevention and treatment of filovirus infection in humans. The recent 2013-2015 West African Ebola epidemic underscores the urgency to develop antiviral therapeutics against these infectious diseases. Our previous study showed that GPCR antagonists, particularly histamine receptor antagonists (antihistamines) inhibit Ebola and Marburg virus entry. In this study, we screened a library of 1220 small molecules with predicted antihistamine activity, identified multiple compounds with potent inhibitory activity against entry of both Ebola and Marburg viruses in human cancer cell lines, and confirmed their anti-Ebola activity in human primary cells. These small molecules target a late-stage of Ebola virus entry. Further structure-activity relationship studies around one compound (cp19) reveal the importance of the coumarin fused ring structure, especially the hydrophobic substituents at positions 3 and/or 4, for its antiviral activity, and this identified scaffold represents a favorable starting point for the rapid development of anti-filovirus therapeutic agents. Copyright © 2017 Elsevier B.V. All rights reserved.

A two-step non-flowcytometry-based naïve B cell isolation method and its application in Staphylococcal enterotoxin B (SEB) presentation.

PubMed

Chokeshai-u-saha, Kaj; Buranapraditkun, Supranee; Jacquet, Alain; Nguyen, Catherine; Ruxrungtham, Kiat

2012-09-01

To study the role of human naïve B cells in antigen presentation and stimulation to naïve CD4+ T cell, a suitable method to reproducibly isolate sufficient naïve B cells is required. To improve the purity of isolated naive B cells obtained from a conventional one-step magnetic bead method, we added a rosetting step to enrich total B cell isolates from human whole blood samples prior to negative cell sorting by magnetic beads. The acquired naïve B cells were analyzed for phenotypes and for their role in Staphylococcal enterotoxin B (SEB) presentation to naïve CD4+ T cells. The mean (SD) naïve B cell (CD19+/CD27-) purity obtained from this two-step method compared with the one-step method was 97% (1.0) versus 90% (1.2), respectively. This two-step method can be used with a sample of whole blood as small as 10 ml. The isolated naive B cells were phenotypically at a resting state and were able to prime naïve CD4+ T cell activation by Staphylococcal enterotoxin B (SEB) presentation. This two-step non-flow cytometry-based approach improved the purity of isolated naïve B cells compared with conventional one-step magnetic bead method. It also worked well with a small blood volume. In addition, this study showed that the isolated naïve B cells can present a super-antigen "SEB" to activate naïve CD4 cells. These methods may thus be useful for further in vitro characterization of human naïve B cells and their roles as antigen presenting cells in various diseases.

A Novel Inhibitor Of Topoisomerase I is Selectively Toxic For A Subset of Non-Small Cell Lung Cancer Cell Lines | Office of Cancer Genomics

Cancer.gov

SW044248, identified through a screen for chemicals that are selectively toxic for NSCLC cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was non-toxic to immortalized human bronchial cell lines.

Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

PubMed Central

Wada, Tamaki; Honda, Makoto; Minami, Itsunari; Tooi, Norie; Amagai, Yuji; Nakatsuji, Norio; Aiba, Kazuhiro

2009-01-01

Background There are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods. Methods/Principal Findings We aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment. Conclusions and Significance The series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved. PMID:19701462

Why are breast cancer stem cells resistant to radiation?

DTIC Science & Technology

2013-03-01

of Human Hsp27 in Rodent Cells: Absence of Compensatory Regulation between Small Heat Shock Proteins. J. Therm. Biol., 21, 365-372, 1996. 69...Corry, P.M., and Lee, Y.J. Comparison of Tumor Growth between Hsp25 and Hsp27 Transfected Murine L929 Cells in Nude Mice. Int. J. Cancer, 72, 871

Silencing the Snail-dependent RNA splice regulator ESRP1 drives malignant transformation of human pulmonary epithelial cells. | Office of Cancer Genomics

Cancer.gov

Epithelial-to-mesenchymal transition (EMT) is organized in cancer cells by a set of key transcription factors, but the significance of this process is still debated including in non-small cell lung cancer (NSCLC). Here we report increased expression of the EMT-inducing transcription factor Snail in premalignant pulmonary lesions, relative to histologically normal pulmonary epithelium. In immortalized human pulmonary epithelial cells and isogenic derivatives, we documented Snail-dependent anchorage-independent growth in vitro and primary tumor growth and metastatic behavior in vivo.

Albumin nanocapsules containing fenretinide: pre-clinical evaluation of cytotoxic activity in experimental models of human non-small cell lung cancer.

PubMed

Pignatta, Sara; Orienti, Isabella; Falconi, Mirella; Teti, Gabriella; Arienti, Chiara; Medri, Laura; Zanoni, Michele; Carloni, Silvia; Zoli, Wainer; Amadori, Dino; Tesei, Anna

2015-02-01

The present study deals with the preparation of albumin nanocapsules containing fenretinide and their evaluation in experimental models of human non-small cell lung cancer. These nanocapsules showed enhanced antitumor activity with respect to free fenretinide due to the solubilization effect of albumin on the hydrophobic drug, known to improve bioavailability. The high expression of caveolin-1 on the A549 cell surface further enhanced the antitumor activity of the nanoencapsulated fenretinide. Caveolin-1 favored albumin uptake and improved the efficacy of the fenretinide-loaded albumin nanocapsules, especially in 3-D cultures where the densely packed 3-D structures impaired drug diffusibility and severely reduced the activity of the free drug. The efficacy of the fenretinide albumin nanocapsules was further confirmed in tumor xenograft models of A549 by the significant delay in tumor progression observed with respect to control after intravenous administration of the novel formulation. This study describes the preparation of fenretinide containing albumin nanocapsules and their evaluation in experimental models of non-small cell lung cancer, showing enhanced antitumor activity compared to free fenretinide. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

PubMed Central

Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

2016-01-01

Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines. PMID:27589719

Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

PubMed

Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

2016-08-29

Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells.

PubMed

Du, Zhong-Wei; Chen, Hong; Liu, Huisheng; Lu, Jianfeng; Qian, Kun; Huang, CindyTzu-Ling; Zhong, Xiaofen; Fan, Frank; Zhang, Su-Chun

2015-03-25

Human pluripotent stem cells (hPSCs) have opened new opportunities for understanding human development, modelling disease processes and developing new therapeutics. However, these applications are hindered by the low efficiency and heterogeneity of cell types, such as motorneurons (MNs), differentiated from hPSCs as well as our inability to maintain the potency of lineage-committed progenitors. Here by using a combination of small molecules that regulate multiple signalling pathways, we develop a method to guide human embryonic stem cells to a near-pure population (>95%) of motor neuron progenitors (MNPs) in 12 days, and an enriched population (>90%) of functionally mature MNs in an additional 16 days. More importantly, the MNPs can be expanded for at least five passages so that a single MNP can be amplified to 1 × 10(4). This method is reproducible in human-induced pluripotent stem cells and is applied to model MN-degenerative diseases and in proof-of-principle drug-screening assays.

Hydrolytic Fate of 3/15-Acetyldeoxynivalenol in Humans: Specific Deacetylation by the Small Intestine and Liver Revealed Using in Vitro and ex Vivo Approaches

PubMed Central

Ajandouz, El Hassan; Berdah, Stéphane; Moutardier, Vincent; Bege, Thierry; Birnbaum, David Jérémie; Perrier, Josette; Di Pasquale, Eric; Maresca, Marc

2016-01-01

In addition to deoxynivalenol (DON), acetylated derivatives, i.e., 3-acetyl and 15-acetyldexynivalenol (or 3/15ADON), are present in cereals leading to exposure to these mycotoxins. Animal and human studies suggest that 3/15ADON are converted into DON after their ingestion through hydrolysis of the acetyl moiety, the site(s) of such deacetylation being still uncharacterized. We used in vitro and ex vivo approaches to study the deacetylation of 3/15ADON by enzymes and cells/tissues present on their way from the food matrix to the blood in humans. We found that luminal deacetylation by digestive enzymes and bacteria is limited. Using human cells, tissues and S9 fractions, we were able to demonstrate that small intestine and liver possess strong deacetylation capacity compared to colon and kidneys. Interestingly, in most cases, deacetylation was more efficient for 3ADON than 15ADON. Although we initially thought that carboxylesterases (CES) could be responsible for the deacetylation of 3/15ADON, the use of pure human CES1/2 and of CES inhibitor demonstrated that CES are not involved. Taken together, our original model system allowed us to identify the small intestine and the liver as the main site of deacetylation of ingested 3/15ADON in humans. PMID:27483321

Induced pluripotent stem (iPS) cells from human fetal stem cells.

PubMed

Guillot, Pascale V

2016-02-01

Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells.

PubMed

Kondo, Yasushi; Toyoda, Taro; Ito, Ryo; Funato, Michinori; Hosokawa, Yoshiya; Matsui, Satoshi; Sudo, Tomomi; Nakamura, Masahiro; Okada, Chihiro; Zhuang, Xiaotong; Watanabe, Akira; Ohta, Akira; Inagaki, Nobuya; Osafune, Kenji

2017-08-01

Pancreatic beta-like cells generated from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) offer an appealing donor tissue source. However, differentiation protocols that mainly use growth factors are costly. Therefore, in this study, we aimed to establish efficient differentiation protocols to change hiPSCs/hESCs to insulin (INS) + cells using novel small-molecule inducers. We screened small molecules that increased the induction rate of INS + cells from hESC-derived pancreatic and duodenal homeobox 1 (PDX1) + pancreatic progenitor cells. The differentiation protocol to generate INS + cells from hiPSCs/hESCs was optimised using hit compounds, and INS + cells induced with the compounds were characterised for their in vitro and in vivo functions. The inducing activity of the hit compounds was also examined using mouse embryonic pancreatic tissues in an explant culture system. Finally, RNA sequencing analyses were performed on the INS + cells to elucidate the mechanisms of action by which the hit compounds induced pancreatic endocrine differentiation. One hit compound, sodium cromoglicate (SCG), was identified out of approximately 1250 small molecules screened. When SCG was combined with a previously described protocol, the induction rate of INS + cells increased from a mean ± SD of 5.9 ± 1.5% (n = 3) to 16.5 ± 2.1% (n = 3). SCG induced neurogenin 3-positive cells at a mean ± SD of 32.6 ± 4.6% (n = 3) compared with 14.2 ± 3.6% (n = 3) for control treatment without SCG, resulting in an increased generation of endocrine cells including insulin-producing cells. Similar induction by SCG was confirmed using mouse embryonic pancreatic explants. We also confirmed that the mechanisms of action by which SCG induced pancreatic endocrine differentiation included the inhibition of bone morphogenetic protein 4 signalling. SCG improves the generation of pancreatic endocrine cells from multiple hiPSC/hESC lines and mouse embryonic pancreatic explants by facilitating the differentiation of endocrine precursors. This discovery will contribute to elucidating the mechanisms of pancreatic endocrine development and facilitate cost-effective generation of INS + cells from hiPSCs/hESCs. The RNA sequencing data generated during the current study are available in the Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo ) with series accession number GSE89973.

Testing lung cancer drugs and therapies in mice

Cancer.gov

National Cancer Institute (NCI) investigators have designed a genetically engineered mouse for use in the study of human lung squamous cell carcinoma (SCC). SCC is a type of non-small cell lung carcinoma, one of the most common types of lung cancer, with

Metabolomics of Small Numbers of Cells: Metabolomic Profiling of 100, 1000, and 10000 Human Breast Cancer Cells.

PubMed

Luo, Xian; Li, Liang

2017-11-07

In cellular metabolomics, it is desirable to carry out metabolomic profiling using a small number of cells in order to save time and cost. In some applications (e.g., working with circulating tumor cells in blood), only a limited number of cells are available for analysis. In this report, we describe a method based on high-performance chemical isotope labeling (CIL) nanoflow liquid chromatography mass spectrometry (nanoLC-MS) for high-coverage metabolomic analysis of small numbers of cells (i.e., ≤10000 cells). As an example, 12 C-/ 13 C-dansyl labeling of the metabolites in lysates of 100, 1000, and 10000 MCF-7 breast cancer cells was carried out using a new labeling protocol tailored to handle small amounts of metabolites. Chemical-vapor-assisted ionization in a captivespray interface was optimized for improving metabolite ionization and increasing robustness of nanoLC-MS. Compared to microflow LC-MS, the nanoflow system provided much improved metabolite detectability with a significantly reduced sample amount required for analysis. Experimental duplicate analyses of biological triplicates resulted in the detection of 1620 ± 148, 2091 ± 89 and 2402 ± 80 (n = 6) peak pairs or metabolites in the amine/phenol submetabolome from the 12 C-/ 13 C-dansyl labeled lysates of 100, 1000, and 10000 cells, respectively. About 63-69% of these peak pairs could be either identified using dansyl labeled standard library or mass-matched to chemical structures in human metabolome databases. We envisage the routine applications of this method for high-coverage quantitative cellular metabolomics using a starting material of 10000 cells. Even for analyzing 100 or 1000 cells, although the metabolomic coverage is reduced from the maximal coverage, this method can still detect thousands of metabolites, allowing the analysis of a large fraction of the metabolome and focused analysis of the detectable metabolites.

CD200-expressing human basal cell carcinoma cells initiate tumor growth.

PubMed

Colmont, Chantal S; Benketah, Antisar; Reed, Simon H; Hawk, Nga V; Telford, William G; Ohyama, Manabu; Udey, Mark C; Yee, Carole L; Vogel, Jonathan C; Patel, Girish K

2013-01-22

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.

Establishment and transformation of telomerase-immortalized human small airway epithelial cells by heavy ions

NASA Astrophysics Data System (ADS)

Zhao, Y. L.; Piao, C. Q.; Hei, T. K.

Previous studies from this laboratory have identified a number of causally linked genes including the novel tumor suppressor Betaig-h3 that were differentially expressed in radiation induced tumorigenic BEP2D cells. To extend these studies using a genomically more stable bronchial cell line, we show here that ectopic expression of the catalytic subunit of telomerase (hTERT) in primary human small airway epithelial (SAE) cells resulted in the generation of several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal. Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings. The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice. These cells show no alteration in the p53 gene but a decrease in p16 expression. Exponentially growing SAEh cells were exposed to graded doses of 1 GeV/nucleon of 56Fe ions accelerated at the Brookhaven National Laboratory. Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation. Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium. These findings indicate that hTERT-immortalized cells, being diploid and chromosomal stable, should be a useful model in assessing mechanism of radiation carcinogenesis.

Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development

PubMed Central

Mora-Bermúdez, Felipe; Badsha, Farhath; Kanton, Sabina; Camp, J Gray; Vernot, Benjamin; Köhler, Kathrin; Voigt, Birger; Okita, Keisuke; Maricic, Tomislav; He, Zhisong; Lachmann, Robert; Pääbo, Svante; Treutlein, Barbara; Huttner, Wieland B

2016-01-01

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. DOI: http://dx.doi.org/10.7554/eLife.18683.001 PMID:27669147

Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies.

PubMed

Friedman, Jay; Morisada, Megan; Sun, Lillian; Moore, Ellen C; Padget, Michelle; Hodge, James W; Schlom, Jeffrey; Gameiro, Sofia R; Allen, Clint T

2018-06-21

Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined. Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed. Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab. Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study.

PubMed

Asgeirsdóttir, Sigridur A; Talman, Eduard G; de Graaf, Inge A; Kamps, Jan A A M; Satchell, Simon C; Mathieson, Peter W; Ruiters, Marcel H J; Molema, Grietje

2010-01-25

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing. Copyright 2009 Elsevier B.V. All rights reserved.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells.

PubMed

Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2015-01-09

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells

PubMed Central

Yang, Jianhong; Zhou, Yongzhao; Cheng, Xia; Fan, Yi; He, Shichao; Li, Shucai; Ye, Haoyu; Xie, Caifeng; Wu, Wenshuang; Li, Chunyan; Pei, Heying; Li, Luyuan; Wei, Zhe; Peng, Aihua; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2015-01-01

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting. PMID:25571970

Metabolic profiling of human lung cancer blood plasma using 1H NMR spectroscopy

NASA Astrophysics Data System (ADS)

Kokova, Daria; Dementeva, Natalia; Kotelnikov, Oleg; Ponomaryova, Anastasia; Cherdyntseva, Nadezhda; Kzhyshkowska, Juliya

2017-11-01

Lung cancer (both small cell and non-small cell) is the second most common cancer in both men and women. The article represents results of evaluating of the plasma metabolic profiles of 100 lung cancer patients and 100 controls to investigate significant metabolites using 400 MHz 1H NMR spectrometer. The results of multivariate statistical analysis show that a medium-field NMR spectrometer can obtain the data which are already sufficient for clinical metabolomics.

Comparisons between Murine Polyomavirus and Simian Virus 40 Show Significant Differences in Small T Antigen Function ▿

PubMed Central

Andrabi, Shaida; Hwang, Justin H.; Choe, Jennifer Kean; Roberts, Thomas M.; Schaffhausen, Brian S.

2011-01-01

Although members of a virus family produce similar gene products, those products may have quite different functions. Simian virus 40 (SV40) large T antigen (LT), for example, targets p53 directly, but murine polyomavirus LT does not. SV40 small T antigen (SVST) has received considerable attention because of its ability to contribute to transformation of human cells. Here, we show that there are major differences between SVST and polyomavirus small T antigen (POLST) in their effects on differentiation, transformation, and cell survival. Both SVST and POLST induce cell cycle progression. However, POLST also inhibits differentiation of 3T3-L1 preadipocytes and C2C12 myoblasts. Additionally, POLST induces apoptosis of mouse embryo fibroblasts. SVST reduces the proapoptotic transcriptional activity of FOXO1 through phosphorylation. On the other hand, SVST complements large T antigen and Ras for the transformation of human mammary epithelial cells (HMECs), but POLST does not. Mechanistically, the differences between SVST and POLST may lie in utilization of protein phosphatase 2A (PP2A). POLST binds both Aα and Aβ scaffolding subunits of PP2A while SVST binds only Aα. Knockdown of Aβ could mimic POLST-induced apoptosis. The two small T antigens can target different proteins for dephosphorylation. POLST binds and dephosphorylates substrates, such as lipins, that SVST does not. PMID:21835797

Engineered kinesin motor proteins amenable to small-molecule inhibition

PubMed Central

Engelke, Martin F.; Winding, Michael; Yue, Yang; Shastry, Shankar; Teloni, Federico; Reddy, Sanjay; Blasius, T. Lynne; Soppina, Pushpanjali; Hancock, William O.; Gelfand, Vladimir I.; Verhey, Kristen J.

2016-01-01

The human genome encodes 45 kinesin motor proteins that drive cell division, cell motility, intracellular trafficking and ciliary function. Determining the cellular function of each kinesin would benefit from specific small-molecule inhibitors. However, screens have yielded only a few specific inhibitors. Here we present a novel chemical-genetic approach to engineer kinesin motors that can carry out the function of the wild-type motor yet can also be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we develop two independent strategies to generate inhibitable motors, and characterize the resulting inhibition in single-molecule assays and in cells. We further apply these two strategies to create analogously inhibitable kinesin-3 motors. These inhibitable motors will be of great utility to study the functions of specific kinesins in a dynamic manner in cells and animals. Furthermore, these strategies can be used to generate inhibitable versions of any motor protein of interest. PMID:27045608

The CXCR4/SDF-1 chemokine receptor axis: a new target therapeutic for non-small cell lung cancer.

PubMed

Otsuka, Shannon; Bebb, Gwyn

2008-12-01

Chemokines are proinflammatory chemoattractant cytokines that regulate cell trafficking and adhesion. The CXCR4 chemokine receptor and its ligand, stromal cell derived factor (SDF-1), constitute a chemokine/receptor axis that has attracted great interest because of an increasing understanding of its role in cancer, including lung cancer. The CXCR4/SDF-1 complex activates several pathways that mediate chemotaxis, migration and secretion of angiopoietic factors. Neutralization of SDF-1 by anti-SDF-1 or anti-CXCR4 monoclonal antibody in preclinical in vivo studies results in a significant decrease of non-small cell lung cancer metastases. Since anti-SDF-1/CXCR4 strategies have already been developed for use in combating human immunodeficiency virus infections, it is likely that these approaches will be used in clinical trials in non-small cell lung cancer in the very near future.

SIRT1 deacetylase is overexpressed in human melanoma and its small molecule inhibition imparts anti-proliferative response via p53 activation.

PubMed

Wilking, Melissa J; Singh, Chandra; Nihal, Minakshi; Zhong, Weixiong; Ahmad, Nihal

2014-12-01

Melanoma causes more deaths than any other skin cancer, and its incidence in the US continues to rise. Current medical therapies are insufficient to control this deadly neoplasm, necessitating the development of new target-based approaches. The objective of this study was to determine the role and functional significance of the class III histone deacetylase SIRT1 in melanoma. We have found that SIRT1 is overexpressed in clinical human melanoma tissues and human melanoma cell lines (Sk-Mel-2, WM35, G361, A375, and Hs294T) compared to normal skin and normal melanocytes, respectively. In addition, treatment of melanoma cell lines A375, Hs294T, and G361 with Tenovin-1, a small molecule SIRT1 inhibitor, resulted in a significant decrease in cell growth and cell viability. Further, Tenovin-1 treatment also resulted in a marked decrease in the clonogenic survival of melanoma cells. Further experiments showed that the anti-proliferative response of Tenovin-1 was accompanied by an increase in the protein as well as activity of the tumor suppressor p53. This increase in p53 activity was substantiated by an increase in the protein level of its downstream target p21. Overall, these data suggest that small molecule inhibition of SIRT1 causes anti-proliferative effects in melanoma cells. SIRT1 appears to be acting through the activity of the tumor suppressor p53, which is not mutated in the majority of melanomas. However, future detailed studies are needed to further explore the role and mechanism of SIRT1 in melanoma development and progression and its usefulness in melanoma treatment.

Chlorella sorokiniana induces mitochondrial-mediated apoptosis in human non-small cell lung cancer cells and inhibits xenograft tumor growth in vivo.

PubMed

Lin, Ping-Yi; Tsai, Ching-Tsan; Chuang, Wan-Ling; Chao, Ya-Hsuan; Pan, I-Horng; Chen, Yu-Kuo; Lin, Chi-Chen; Wang, Bing-Yen

2017-02-01

Lung cancer is one of the leading causes of cancer related deaths worldwide. Marine microalgae are a source of biologically active compounds and are widely consumed as a nutritional supplement in East Asian countries. It has been reported that Chlorella or Chlorella extracts have various beneficial pharmacological compounds that modulate immune responses; however, no studies have investigated the anti-cancer effects of Chlorella sorokiniana (CS) on non-small cell lung cancer (NSCLC). In this study, we evaluated the anti-cancer effects of CS in two human NSCLC cell lines (A549 and CL1-5 human lung adenocarcinoma cells), and its effects on tumor growth in a subcutaneous xenograft tumor model. We also investigated the possible molecular mechanisms governing the pharmacological function of CS. Our results showed that exposure of the two cell lines to CS resulted in a concentration-dependent reduction in cell viability. In addition, the percentage of apoptotic cells increased in a dose-dependent manner, suggesting that CS might induce apoptosis in human NSCLC cells. Western blot analysis revealed that exposure to CS resulted in increased protein expression of the cleaved/activated forms of caspase-3, caspase-9, and PARP, except caspase-8. ZDEVD (caspase-3 inhibitor) and Z-LEHD (caspase-9 inhibitor) were sufficient at preventing apoptosis in both A549 and CL1-5 cells, proving that CS induced cell death via the mitochondria-mediated apoptotic pathway. Exposure of A549 and CL1-5 cells to CS for 24 h resulted in decreased expression of Bcl-2 protein and increased expression of Bax protein as well as decreased expression of two IAP family proteins, survivin and XIAP. We demonstrated that CS induces mitochondrial-mediated apoptosis in NSCLC cells via downregulation of Bcl-2, XIAP and survivin. In addition, we also found that the tumors growth of subcutaneous xenograft in vivo was markedly inhibited after oral intake of CS.

Potent Cytotoxic Peptides from the Australian Marine Sponge Pipestela candelabra

PubMed Central

Tran, Trong D.; Pham, Ngoc B.; Fechner, Gregory A.; Hooper, John N. A.; Quinn, Ronald J.

2014-01-01

Two consecutive prefractionated fractions of the Australian marine sponge extract, Pipestela candelabra, were identified to be selectively active on the human prostate cancer cells (PC3) compared to the human neonatal foreskin fibroblast non-cancer cells (NFF). Twelve secondary metabolites were isolated in which four compounds are new small peptides. Their structures were characterized by spectroscopic and chemical analysis. These compounds inhibited selectively the growth of prostate cancer cells with IC50 values in the picomolar to sub-micromolar range. Structure-activity relationship of these compounds is discussed. PMID:24901701

A simple method to assess in vivo proliferation in lung vasculature with EdU: the case of MMC-induced PVOD in rat.

PubMed

Fabrice, Antigny; Benoît, Ranchoux; Valérie, Nadeau; Lau, Edmund; Sébastien, Bonnet; Frédéric, Perros

2015-01-01

5-Ethynyl-2'-deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a "terminal" state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD. In vivo pulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.

Arterial and venous endothelia display differential functional fractalkine (CX3CL1) expression by angiotensin-II.

PubMed

Rius, Cristina; Piqueras, Laura; González-Navarro, Herminia; Albertos, Fernando; Company, Chantal; López-Ginés, Concha; Ludwig, Andreas; Blanes, Jose-Ignacio; Morcillo, Esteban J; Sanz, Maria-Jesus

2013-01-01

Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX(3)CL1) was explored. Enhanced CX(3)CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX(3)CL1 receptor (CX(3)CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 μmol/L Ang-II increased CX(3)CL1 expression, yet neutralization of CX(3)CL1 activity only significantly inhibited Ang-II-induced mononuclear cell-human umbilical arterial endothelial cell interactions (73%) but not with human umbilical venous endothelial cells. The use of small interfering RNA revealed the involvement of tumor necrosis factor-α in Ang-II-induced CX(3)CL1 upregulation and mononuclear cell arrest. Nox5 knockdown with small interfering RNA or pharmacological inhibition of extracellular signal-regulated kinases1/2, p38 mitogen-activated protein kinase, and nuclear factor-κB also abolished these responses. Finally, when human umbilical arterial endothelial cells were costimulated with Ang-II, tumor necrosis factor-α, and interferon-γ, CX(3)CL1 expression and mononuclear cell adhesiveness were more pronounced than when each stimulus was provided alone. These results suggest that Ang-II induces functional CX(3)CL1 expression in arterial but not in venous endothelia. Thus, targeting endothelial CX(3)CL1-mononuclear leukocyte CX(3)CR1 interactions may constitute a new therapeutic strategy in the treatment of Ang-II-associated cardiovascular diseases.

Isolation and characterisation of mesenchymal stem/stromal cells in the ovine endometrium.

PubMed

Letouzey, Vincent; Tan, Ker Sin; Deane, James A; Ulrich, Daniela; Gurung, Shanti; Ong, Y Rue; Gargett, Caroline E

2015-01-01

Mesenchymal stem/stromal cells (MSC) were recently discovered in the human endometrium. These cells possess key stem cell properties and show promising results in small animal models when used for preclinical tissue engineering studies. A small number of surface markers have been identified that enrich for MSC from bone marrow and human endometrium, including the Sushi Domain-containing 2 (SUSD2; W5C5) and CD271 markers. In preparation for developing a large animal preclinical model for urological and gynecological tissue engineering applications we aimed to identify and characterise MSC in ovine endometrium and determine surface markers to enable their prospective isolation. Ovine endometrium was obtained from hysterectomised ewes following progesterone synchronisation, dissociated into single cell suspensions and tested for MSC surface markers and key stem cell properties. Purified stromal cells were obtained by flow cytometry sorting with CD49f and CD45 to remove epithelial cells and leukocytes respectively, and MSC properties investigated. There was a small population CD271+ stromal cells (4.5 ± 2.3%) in the ovine endometrium. Double labelling with CD271 and CD49f showed that the sorted CD271+CD49f- stromal cell population possessed significantly higher cloning efficiency, serial cloning capacity and a qualitative increased ability to differentiate into 4 mesodermal lineages (adipocytic, smooth muscle, chondrocytic and osteoblastic) than CD271-CD49f- cells. Immunolabelling studies identified an adventitial perivascular location for ovine endometrial CD271+ cells. This is the first study to characterise MSC in the ovine endometrium and identify a surface marker profile identifying their location and enabling their prospective isolation. This knowledge will allow future preclinical studies with a large animal model that is well established for pelvic organ prolapse research.

Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis.

PubMed

Yang, Yi; Zhao, Yi; Ai, Xinghao; Cheng, Baijun; Lu, Shun

2014-01-01

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells.

Human reaming debris: a source of multipotent stem cells.

PubMed

Wenisch, Sabine; Trinkaus, Katja; Hild, Anne; Hose, Dirk; Herde, Katja; Heiss, Christian; Kilian, Olaf; Alt, Volker; Schnettler, Reinhard

2005-01-01

The biological characteristics of human reaming debris (HRD) generated in the course of surgical treatment of long bone diaphyseal fractures and nonunions are still a matter of dispute. Therefore, the objective of the present investigation has been to characterize the intrinsic properties of human reaming debris in vitro. Samples of reaming debris harvested from 12 patients with closed diaphyseal fractures were examined ultrastucturally and were cultured under standard conditions. After a lag phase of 4-7 days, cells started to grow out from small bone fragments and established a confluent monolayer within 20-22 days. The cells were characterized according to morphology, proliferation capacity, cell surface antigen profile, and differentiation repertoire. The results reveal that human reaming debris is a source of multipotent stem cells which are able to grow and proliferate in vitro. The cells differentiate along the osteogenic pathway after induction and can be directed toward a neuronal phenotype, as has been shown morphologically and by the expression of neuronal markers after DMSO induction. These findings have prompted interest in the use of reaming debris-derived stem cells in cell and bone replacement therapies.

Antiproliferative and apoptotic effects of chamomile extract in various human cancer cells.

PubMed

Srivastava, Janmejai K; Gupta, Sanjay

2007-11-14

Chamomile (Matricaria chamomilla), a popular herb valued for centuries as a traditional medicine, has been used to treat various human ailments; however, its anticancer activity is unknown. We evaluated the anticancer properties of aqueous and methanolic extracts of chamomile against various human cancer cell lines. Exposure of chamomile extracts caused minimal growth inhibitory responses in normal cells, whereas a significant decrease in cell viability was observed in various human cancer cell lines. Chamomile exposure resulted in differential apoptosis in cancer cells but not in normal cells at similar doses. HPLC analysis of chamomile extract confirmed apigenin 7-O-glucoside as the major constituent of chamomile; some minor glycoside components were also observed. Apigenin glucosides inhibited cancer cell growth but to a lesser extent than the parent aglycone, apigenin. Ex vivo experiments suggest that deconjugation of glycosides occurs in vivo to produce aglycone, especially in the small intestine. This study represents the first reported demonstration of the anticancer effects of chamomile. Further investigations of the mechanism of action of chamomile are warranted in evaluating the potential usefulness of this herbal remedy in the management of cancer patients.

SPAR: small RNA-seq portal for analysis of sequencing experiments.

PubMed

Kuksa, Pavel P; Amlie-Wolf, Alexandre; Katanic, Živadin; Valladares, Otto; Wang, Li-San; Leung, Yuk Yee

2018-05-04

The introduction of new high-throughput small RNA sequencing protocols that generate large-scale genomics datasets along with increasing evidence of the significant regulatory roles of small non-coding RNAs (sncRNAs) have highlighted the urgent need for tools to analyze and interpret large amounts of small RNA sequencing data. However, it remains challenging to systematically and comprehensively discover and characterize sncRNA genes and specifically-processed sncRNA products from these datasets. To fill this gap, we present Small RNA-seq Portal for Analysis of sequencing expeRiments (SPAR), a user-friendly web server for interactive processing, analysis, annotation and visualization of small RNA sequencing data. SPAR supports sequencing data generated from various experimental protocols, including smRNA-seq, short total RNA sequencing, microRNA-seq, and single-cell small RNA-seq. Additionally, SPAR includes publicly available reference sncRNA datasets from our DASHR database and from ENCODE across 185 human tissues and cell types to produce highly informative small RNA annotations across all major small RNA types and other features such as co-localization with various genomic features, precursor transcript cleavage patterns, and conservation. SPAR allows the user to compare the input experiment against reference ENCODE/DASHR datasets. SPAR currently supports analyses of human (hg19, hg38) and mouse (mm10) sequencing data. SPAR is freely available at https://www.lisanwanglab.org/SPAR.

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

PubMed Central

Ravi, R K; Weber, E; McMahon, M; Williams, J R; Baylin, S; Mal, A; Harter, M L; Dillehay, L E; Claudio, P P; Giordano, A; Nelkin, B D; Mabry, M

1998-01-01

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors. PMID:9421477

Replication of Many Human Viruses Is Refractory to Inhibition by Endogenous Cellular MicroRNAs

PubMed Central

Bogerd, Hal P.; Skalsky, Rebecca L.; Kennedy, Edward M.; Furuse, Yuki; Whisnant, Adam W.; Flores, Omar; Schultz, Kimberly L. W.; Putnam, Nicole; Barrows, Nicholas J.; Sherry, Barbara; Scholle, Frank; Garcia-Blanco, Mariano A.; Griffin, Diane E.

2014-01-01

ABSTRACT The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines. PMID:24807715

Proteome-wide covalent ligand discovery in native biological systems

PubMed Central

Backus, Keriann M.; Correia, Bruno E.; Lum, Kenneth M.; Forli, Stefano; Horning, Benjamin D.; González-Páez, Gonzalo E.; Chatterjee, Sandip; Lanning, Bryan R.; Teijaro, John R.; Olson, Arthur J.; Wolan, Dennis W.; Cravatt, Benjamin F.

2016-01-01

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered “undruggable” 1,2. Fragment-based ligand discovery (FBLD) can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries 1,3. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes 4–10, including those that can access regions of proteins that are difficult to access through binding affinity alone 5,10,11. In this manuscript, we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T-cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and −10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems. PMID:27309814

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

PubMed

Malik, Nasir; Efthymiou, Anastasia G; Mather, Karly; Chester, Nathaniel; Wang, Xiantao; Nath, Avindra; Rao, Mahendra S; Steiner, Joseph P

2014-12-01

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens. Published by Elsevier B.V.

RNAi Screening with Self-Delivering, Synthetic siRNAs for Identification of Genes That Regulate Primary Human T Cell Migration.

PubMed

Freeley, Michael; Derrick, Emily; Dempsey, Eugene; Hoff, Antje; Davies, Anthony; Leake, Devin; Vermeulen, Annaleen; Kelleher, Dermot; Long, Aideen

2015-09-01

Screening of RNA interference (RNAi) libraries in primary T cells is labor-intensive and technically challenging because these cells are hard to transfect. Chemically modified, self-delivering small interfering RNAs (siRNAs) offer a solution to this problem, because they enter hard-to-transfect cell types without needing a delivery reagent and are available in library format for RNAi screening. In this study, we have screened a library of chemically modified, self-delivering siRNAs targeting the expression of 72 distinct genes in conjunction with an image-based high-content-analysis platform as a proof-of-principle strategy to identify genes involved in lymphocyte function-associated antigen-1 (LFA-1)-mediated migration in primary human T cells. Our library-screening strategy identified the small GTPase RhoA as being crucial for T cell polarization and migration in response to LFA-1 stimulation and other migratory ligands. We also demonstrate that multiple downstream assays can be performed within an individual RNAi screen and have used the remainder of the cells for additional assays, including cell viability and adhesion to ICAM-1 (the physiological ligand for LFA-1) in the absence or presence of the chemokine SDF-1α. This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function. © 2015 Society for Laboratory Automation and Screening.

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

NASA Technical Reports Server (NTRS)

Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

2005-01-01

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

Unscheduled DNA synthesis in human bronchial epithelium treated with various chemical carcinogens in vitro

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

Ishikawa, T.; Ide, F.; Kodama, K.

1984-07-01

A system was developed in which organ culture of human bronchial epithelium was used in combination with autoradiography for quantitative measurement of unscheduled DNA synthesis (UDS) in bronchial epithelial cells. Human bronchi obtained at surgery were cut into small sections and treated with various carcinogens plus (methyl-/sup 3/H)thymidine in short-term organ culture. Significant numbers of silver grains, indicating UDS, were detected on the nuclei of epithelial cells of human bronchi treated with carcinogens, and the numbers were proportional to the concentrations of carcinogens. In this system seven representative carcinogens induced UDS. Four active metabolites of benzo(a)pyrene, and benz(a)anthracene also weremore » found to induce very active UDS in human bronchial epithelium. These findings suggest that human bronchial epithelial cells can repair different types of DNA modification induced by chemical carcinogens.« less

Exosomal microRNA Biomarkers: Emerging Frontiers in Colorectal and Other Human Cancers

PubMed Central

Goel, Ajay; Tovar-Camargo, Oscar A; Toden, Shusuke

2016-01-01

Diagnostic strategies, particularly non-invasive blood-based screening approaches, are gaining increased attention for the early detection and attenuation of mortality associated with colorectal cancer (CRC). However, the majority of current screening approaches are inadequate at replacing the conventional CRC diagnostic procedures. Yet, due to technological advances and a better understanding of molecular events underlying human cancer, a new category of biomarkers are on the horizon. Recent evidence indicates that cells release a distinct class of small vesicles called ‘exosomes’, which contain nucleic acids and proteins that reflect and typify host-cell molecular architecture. Intriguingly, exosomes released from cancer cells have a distinct genetic and epigenetic makeup, which allows them to undertake their tumorigenic function. From a clinical standpoint, these unique cancer-specific fingerprints present in exosomes appear to be detectable in a small amount of blood, making them very attractive substrates for developing cancer biomarkers, particularly noninvasive diagnostic approaches. PMID:26892862

Activity of a new nitrosourea (TCNU) in human lung cancer xenografts.

PubMed Central

Fergusson, R. J.; Anderson, L. E.; Macpherson, J. S.; Robins, P.; Smyth, J. F.

1988-01-01

The activity of a new nitrosourea (TCNU) based on the endogenous amino acid taurine was assessed in three human lung cancer xenografts growing in immunodeficient mice. Moderate activity (specific growth delays of 0.63 and 1.13 compared with controls) was seen in two non-small cell tumours after a single oral administration of 20 mg-1kg. This dose was curative in a small cell xenograft. By using high performance liquid chromatography it was possible to detect parent drug in the tumours as well as the plasma and tissues after oral administration of TCNU. Drug sensitivity was correlated inversely with the amount of the DNA repair enzyme 0(6)-methylguanine-DNA methyltransferase assayed from extracts of the tumour cells but not with the levels of parent drug within the tumour. This compound appears to have unique pharmacokinetic properties compared with other chloroethylnitrosoureas. PMID:3390369

L1000CDS2: LINCS L1000 characteristic direction signatures search engine.

PubMed

Duan, Qiaonan; Reid, St Patrick; Clark, Neil R; Wang, Zichen; Fernandez, Nicolas F; Rouillard, Andrew D; Readhead, Ben; Tritsch, Sarah R; Hodos, Rachel; Hafner, Marc; Niepel, Mario; Sorger, Peter K; Dudley, Joel T; Bavari, Sina; Panchal, Rekha G; Ma'ayan, Avi

2016-01-01

The library of integrated network-based cellular signatures (LINCS) L1000 data set currently comprises of over a million gene expression profiles of chemically perturbed human cell lines. Through unique several intrinsic and extrinsic benchmarking schemes, we demonstrate that processing the L1000 data with the characteristic direction (CD) method significantly improves signal to noise compared with the MODZ method currently used to compute L1000 signatures. The CD processed L1000 signatures are served through a state-of-the-art web-based search engine application called L1000CDS 2 . The L1000CDS 2 search engine provides prioritization of thousands of small-molecule signatures, and their pairwise combinations, predicted to either mimic or reverse an input gene expression signature using two methods. The L1000CDS 2 search engine also predicts drug targets for all the small molecules profiled by the L1000 assay that we processed. Targets are predicted by computing the cosine similarity between the L1000 small-molecule signatures and a large collection of signatures extracted from the gene expression omnibus (GEO) for single-gene perturbations in mammalian cells. We applied L1000CDS 2 to prioritize small molecules that are predicted to reverse expression in 670 disease signatures also extracted from GEO, and prioritized small molecules that can mimic expression of 22 endogenous ligand signatures profiled by the L1000 assay. As a case study, to further demonstrate the utility of L1000CDS 2 , we collected expression signatures from human cells infected with Ebola virus at 30, 60 and 120 min. Querying these signatures with L1000CDS 2 we identified kenpaullone, a GSK3B/CDK2 inhibitor that we show, in subsequent experiments, has a dose-dependent efficacy in inhibiting Ebola infection in vitro without causing cellular toxicity in human cell lines. In summary, the L1000CDS 2 tool can be applied in many biological and biomedical settings, while improving the extraction of knowledge from the LINCS L1000 resource.

Expression of TMPRSS4 in non-small cell lung cancer and its modulation by hypoxia

PubMed Central

NGUYEN, TRI-HUNG; WEBER, WILLIAM; HAVARI, EVIS; CONNORS, TIMOTHY; BAGLEY, REBECCA G.; McLAREN, RAJASHREE; NAMBIAR, PRASHANT R.; MADDEN, STEPHEN L.; TEICHER, BEVERLY A.; ROBERTS, BRUCE; KAPLAN, JOHANNE; SHANKARA, SRINIVAS

2012-01-01

Overexpression of TMPRSS4, a cell surface-associated transmembrane serine protease, has been reported in pancreatic, colorectal and thyroid cancers, and has been implicated in tumor cell migration and metastasis. Few reports have investigated both TMPRSS4 gene expression levels and the protein products. In this study, quantitative RT-PCR and protein staining were used to assess TMPRSS4 expression in primary non-small cell lung carcinoma (NSCLC) tissues and in lung tumor cell lines. At the transcriptional level, TMPRSS4 message was significantly elevated in the majority of human squamous cell and adenocarcinomas compared with normal lung tissues. Staining of over 100 NSCLC primary tumor and normal specimens with rabbit polyclonal anti-TMPRSS4 antibodies confirmed expression at the protein level in both squamous cell and adenocarcinomas with little or no staining in normal lung tissues. Human lung tumor cell lines expressed varying levels of TMPRSS4 mRNA in vitro. Interestingly, tumor cell lines with high levels of TMPRSS4 mRNA failed to show detectable TMPRSS4 protein by either immunoblotting or flow cytometry. However, protein levels were increased under hypoxic culture conditions suggesting that hypoxia within the tumor microenvironment may upregulate TMPRSS4 protein expression in vivo. This was supported by the observation of TMPRSS4 protein in xenograft tumors derived from the cell lines. In addition, staining of human squamous cell carcinoma samples for carbonic anhydrase IX (CAIX), a hypoxia marker, showed TMPRSS4 positive cells adjacent to CAIX positive cells. Overall, these results indicate that the cancer-associated TMPRSS4 protein is overexpressed in NSCLC and may represent a potential therapeutic target. PMID:22692880

The small tellurium-based compound SAS suppresses inflammation in human retinal pigment epithelium

PubMed Central

Livnat, Tami; Halpert, Gilad; Jawad, Shayma; Nisgav, Yael; Azar-Avivi, Shirley; Liu, Baoying; Nussenblatt, Robert B.; Weinberger, Dov; Sredni, Benjamin

2016-01-01

Purpose Pathological angiogenesis and chronic inflammation greatly contribute to the development of choroidal neovascularization (CNV) in chorioretinal diseases involving abnormal contact between retinal pigment epithelial (RPE) and endothelial cells (ECs), associated with Bruch’s membrane rupture. We explored the ability of the small organotellurium compound octa-O-bis-(R,R)-tartarate ditellurane (SAS) to mitigate inflammatory processes in human RPE cells. Methods Cell adhesion assays and analyses of gene and protein expression were used to examine the effect of SAS on ARPE-19 cells or primary human RPE cells that were grown alone or in an RPE-EC co-culture. Results Adhesion assays showed that SAS inhibited αv integrins expressed on RPE cells. Co-cultures of RPE cells with ECs significantly reduced the gene expression of PEDF, as compared to RPE cells cultured alone. Both SAS and the anti-αvβ3 antibody LM609 significantly enhanced the production of PEDF at both mRNA and protein levels in RPE cells. RPE cells co-cultured with EC exhibited increased gene expression of CXCL5, COX1, MMP2, IGF1, and IL8, all of which are involved in both angiogenesis and inflammation. The enhanced expression of these genes was greatly suppressed by SAS, but interestingly, remained unaffected by LM609. Zymography assay showed that SAS reduced the level of MMP-2 activity in RPE cells. We also found that SAS significantly suppressed IL-1β-induced IL-6 expression and secretion from RPE cells by reducing the protein levels of phospho-IkappaBalpha (pIκBα). Conclusions Our results suggest that SAS is a promising anti-inflammatory agent in RPE cells, and may be an effective therapeutic approach for controlling chorioretinal diseases. PMID:27293373

The small tellurium-based compound SAS suppresses inflammation in human retinal pigment epithelium.

PubMed

Dardik, Rima; Livnat, Tami; Halpert, Gilad; Jawad, Shayma; Nisgav, Yael; Azar-Avivi, Shirley; Liu, Baoying; Nussenblatt, Robert B; Weinberger, Dov; Sredni, Benjamin

2016-01-01

Pathological angiogenesis and chronic inflammation greatly contribute to the development of choroidal neovascularization (CNV) in chorioretinal diseases involving abnormal contact between retinal pigment epithelial (RPE) and endothelial cells (ECs), associated with Bruch's membrane rupture. We explored the ability of the small organotellurium compound octa-O-bis-(R,R)-tartarate ditellurane (SAS) to mitigate inflammatory processes in human RPE cells. Cell adhesion assays and analyses of gene and protein expression were used to examine the effect of SAS on ARPE-19 cells or primary human RPE cells that were grown alone or in an RPE-EC co-culture. Adhesion assays showed that SAS inhibited αv integrins expressed on RPE cells. Co-cultures of RPE cells with ECs significantly reduced the gene expression of PEDF, as compared to RPE cells cultured alone. Both SAS and the anti-αvβ3 antibody LM609 significantly enhanced the production of PEDF at both mRNA and protein levels in RPE cells. RPE cells co-cultured with EC exhibited increased gene expression of CXCL5, COX1, MMP2, IGF1, and IL8, all of which are involved in both angiogenesis and inflammation. The enhanced expression of these genes was greatly suppressed by SAS, but interestingly, remained unaffected by LM609. Zymography assay showed that SAS reduced the level of MMP-2 activity in RPE cells. We also found that SAS significantly suppressed IL-1β-induced IL-6 expression and secretion from RPE cells by reducing the protein levels of phospho-IkappaBalpha (pIκBα). Our results suggest that SAS is a promising anti-inflammatory agent in RPE cells, and may be an effective therapeutic approach for controlling chorioretinal diseases.

Tracing the temporal-spatial transcriptome landscapes of the human fetal digestive tract using single-cell RNA-sequencing.

PubMed

Gao, Shuai; Yan, Liying; Wang, Rui; Li, Jingyun; Yong, Jun; Zhou, Xin; Wei, Yuan; Wu, Xinglong; Wang, Xiaoye; Fan, Xiaoying; Yan, Jie; Zhi, Xu; Gao, Yun; Guo, Hongshan; Jin, Xiao; Wang, Wendong; Mao, Yunuo; Wang, Fengchao; Wen, Lu; Fu, Wei; Ge, Hao; Qiao, Jie; Tang, Fuchou

2018-06-01

The development of the digestive tract is critical for proper food digestion and nutrient absorption. Here, we analyse the main organs of the digestive tract, including the oesophagus, stomach, small intestine and large intestine, from human embryos between 6 and 25 weeks of gestation as well as the large intestine from adults using single-cell RNA-seq analyses. In total, 5,227 individual cells are analysed and 40 cell types clearly identified. Their crucial biological features, including developmental processes, signalling pathways, cell cycle, nutrient digestion and absorption metabolism, and transcription factor networks, are systematically revealed. Moreover, the differentiation and maturation processes of the large intestine are thoroughly investigated by comparing the corresponding transcriptome profiles between embryonic and adult stages. Our work offers a rich resource for investigating the gene regulation networks of the human fetal digestive tract and adult large intestine at single-cell resolution.

Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology

PubMed Central

Saxena, Kapil; Blutt, Sarah E.; Ettayebi, Khalil; Zeng, Xi-Lei; Broughman, James R.; Crawford, Sue E.; Karandikar, Umesh C.; Sastri, Narayan P.; Conner, Margaret E.; Opekun, Antone R.; Graham, David Y.; Qureshi, Waqar; Sherman, Vadim; Foulke-Abel, Jennifer; In, Julie; Kovbasnjuk, Olga; Zachos, Nicholas C.; Donowitz, Mark

2015-01-01

ABSTRACT Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion. IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤5 years of age. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, cellularly diverse, and physiologically relevant epithelial cell cultures that recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions; host and cell type restriction; virus-induced fluid secretion; cell-cell communication within the epithelium; and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures. PMID:26446608

A sensitive ELISA for measuring the adhesion of leukocytic cells to human endothelial cells.

PubMed

Krakauer, T

1994-12-28

A new, sensitive ELISA using monoclonal antibodies reactive with surface molecules specific for various leukocytes was devised to measure the attachment of these cells to cultured monolayers of human umbilical vein endothelial cells. Preparations of peripheral blood mononuclear cells, a human monocytic cell line (THP-1) and a human lymphoblastic T cell line (MOLT-4) were used to test the sensitivity of this method and compare it with the conventional 51Cr-radiolabeled cell assay. The extent of adhesion to endothelial cells was assayed by measuring the optical density produced by a complex of peroxidase-labeled streptavidin, biotin-conjugated F(ab')2 anti-mouse Ig and monoclonal antibody on fixed leukocytic cells that had adhered to endothelial cells. This method is fast and sensitive, eliminates the use of radioisotopes, and, because the detection uses a specific marker on the cell of interest, can be used in preparations of unseparated mixtures of cells. As this is a microassay, using relatively small number of cells and reagents, the methodology can be applied to screen a large number of therapeutic agents that may regulate adhesion. Using this method, the anti-inflammatory corticosteroid, dexamethasone, was found to inhibit the adhesion of THP-1 and MOLT-4 cells to cytokine-activated endothelial cells.

The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

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

Watanabe, H.; Grubb, J.H.; Sly, W.S.

1990-10-01

The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less

High content screening of defined chemical libraries using normal and glioma-derived neural stem cell lines.

PubMed

Danovi, Davide; Folarin, Amos A; Baranowski, Bart; Pollard, Steven M

2012-01-01

Small molecules with potent biological effects on the fate of normal and cancer-derived stem cells represent both useful research tools and new drug leads for regenerative medicine and oncology. Long-term expansion of mouse and human neural stem cells is possible using adherent monolayer culture. These cultures represent a useful cellular resource to carry out image-based high content screening of small chemical libraries. Improvements in automated microscopy, desktop computational power, and freely available image processing tools, now means that such chemical screens are realistic to undertake in individual academic laboratories. Here we outline a cost effective and versatile time lapse imaging strategy suitable for chemical screening. Protocols are described for the handling and screening of human fetal Neural Stem (NS) cell lines and their malignant counterparts, Glioblastoma-derived neural stem cells (GNS). We focus on identification of cytostatic and cytotoxic "hits" and discuss future possibilities and challenges for extending this approach to assay lineage commitment and differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

PubMed

Jiwaji, Meesbah; Short, James Roswell; Dorrington, Rosemary Ann

2016-10-01

Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

Chiral effects in adrenocorticolytic action of o,p'-DDD (mitotane) in human adrenal cells.

PubMed

Asp, V; Cantillana, T; Bergman, A; Brandt, I

2010-03-01

Adrenocortical carcinoma (ACC) is a rare malignant disease with poor prognosis. The main pharmacological choice, o,p'-DDD (mitotane), produces severe adverse effects. Since o,p'-DDD is a chiral molecule and stereoisomers frequently possess different pharmacokinetic and/or pharmacodynamic properties, we isolated the two o,p'-DDD enantiomers, (R)-(+)-o,p'-DDD and (S)-(-)-o,p'-DDD, and determined their absolute structures. The effects of each enantiomer on cell viability and on cortisol and dehydroepiandrosterone (DHEA) secretion in the human adrenocortical cell line H295R were assessed. We also assayed the o,p'-DDD racemate and the m,p'- and p,p'-isomers. The results show small but statistically significant differences in activity of the o,p'-DDD enantiomers for all parameters tested. The three DDD isomers were equally potent in decreasing cell viability, but p,p'-DDD affected hormone secretion slightly less than the o,p'- and m,p'-isomers. The small chiral differences in direct effects on target cells alone do not warrant single enantiomer administration, but might reach importance in conjunction with possible stereochemical effects on pharmacokinetic processes in vivo.

Infrared Hollow Optical Fiber Probe for Localized Carbon Dioxide Measurement in Respiratory Tracts.

PubMed

Katagiri, Takashi; Shibayama, Kyosuke; Iida, Takeru; Matsuura, Yuji

2018-03-27

A real-time gas monitoring system based on optical absorption spectroscopy is proposed for localized carbon dioxide (CO₂) measurement in respiratory tracts. In this system, a small gas cell is attached to the end of a hollow optical fiber that delivers mid-infrared light with small transmission loss. The diameters of the fiber and the gas cell are smaller than 1.2 mm so that the probe can be inserted into a working channel of common bronchoscopes. The dimensions of the gas cell are designed based on absorption spectra of CO₂ standard gases in the 4.2 μm wavelength region, which are measured using a Fourier-transform infrared spectrometer. A miniature gas cell that is comprised of a stainless-steel tube with slots for gas inlet and a micro-mirror is fabricated. A compact probing system with a quantum cascade laser (QCL) light source is built using a gas cell with a hollow optical fiber for monitoring CO₂ concentration. Experimental results using human breaths show the feasibility of the system for in-situ measurement of localized CO₂ concentration in human airways.

Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture

PubMed Central

Takeuchi, Hiroki; Nakatsuji, Norio; Suemori, Hirofumi

2014-01-01

Insulin-producing cells (IPCs) derived from human pluripotent stem cells (hPSCs) may be useful in cell therapy and drug discovery for diabetes. Here, we examined various growth factors and small molecules including those previously reported to develop a robust differentiation method for induction of mature IPCs from hPSCs. We established a protocol that induced PDX1-positive pancreatic progenitor cells at high efficiency, and further induced mature IPCs by treatment with forskolin, dexamethasone, Alk5 inhibitor II and nicotinamide in 3D culture. The cells that differentiated into INSULIN-positive and C-PEPTIDE-positive cells secreted insulin in response to glucose stimulation, indicating a functional IPC phenotype. We also found that this method was applicable to different types of hPSCs. PMID:24671046

JNK signaling mediates EPHA2-dependent tumor cell proliferation, motility, and cancer stem cell-like properties in non-small cell lung cancer

PubMed Central

Song, Wenqiang; Ma, Yufang; Wang, Jialiang; Brantley-Sieders, Dana; Chen, Jin

2014-01-01

Recent genome-wide analyses in human lung cancer revealed that EPHA2 receptor tyrosine kinase is overexpressed in non-small cell lung cancer (NSCLC), and high levels of EPHA2 correlate with poor clinical outcome. However, the mechanistic basis for EPHA2-mediated tumor promotion in lung cancer remains poorly understood. Here we show that the JNK/c-JUN signaling mediates EPHA2-dependent tumor cell proliferation and motility. A screen of phospho-kinase arrays revealed a decrease in phospho-c-JUN levels in EPHA2 knockdown cells. Knockdown of EPHA2 inhibited p-JNK and p-c-JUN levels in approximately 50% of NSCLC lines tested. Treatment of parental cells with SP600125, a JNK inhibitor, recapitulated defects in EPHA2-deficient tumor cells; whereas constitutively activated JNK mutants were sufficient to rescue phenotypes. Knockdown of EPHA2 also inhibited tumor formation and progression in xenograft animal models in vivo. Furthermore, we investigated the role of EPHA2 in cancer stem-like cells. RNAi-mediated depletion of EPHA2 in multiple NSCLC lines decreased the ALDH positive cancer stem-like population and tumor spheroid formation in suspension. Depletion of EPHA2 in sorted ALDH positive populations markedly inhibited tumorigenicity in nude mice. Furthermore, analysis of a human lung cancer tissue microarray revealed a significant, positive association between EPHA2 and ALDH expression, indicating an important role for EPHA2 in human lung cancer stem-like cells. Collectively, these studies revealed a critical role of JNK signaling in EPHA2-dependent lung cancer cell proliferation and motility and a role for EPHA2 in cancer stem-like cell function, providing evidence for EPHA2 as a potential therapeutic target in NSCLC. PMID:24607842

Small interfering RNA-mediated silencing of G-protein-coupled receptor 137 inhibits growth of osteosarcoma cells.

PubMed

Li, Hao; Fu, Xiaodong; Gao, Yingjian; Li, Xiaomiao; Shen, Yi; Wang, Weili

2018-06-01

Osteosarcoma is the most widespread primary carcinoma in bones. Osteosarcoma cells are highly metastatic and frequently develop resistance to chemotherapy making this disease harder to treat. This identifies an urgent need of novel therapeutic strategies for osteosarcoma. G-Protein-coupled receptor 137 (GPR137) is involved in several human cancers and may be a novel therapeutic target. The expression of GPR137 was assessed in one osteoblast and three human osteosarcoma cell lines via the quantitative real-time polymerase chain reaction and western blot assays. Stable GPR137 knockdown cell lines were established using an RNA interference lentivirus system. Viability, colony formation, and flow cytometry assays were performed to measure the effects of GPR137 depletion on cell growth. The underlying molecular mechanism was determined using signaling array analysis and western blot assays. GPR137 expression was higher in the three human osteosarcoma cell lines, Saos-2, U2OS, and SW1353, than in osteoblast hFOB 1.19 cells. Lentivirus-mediated small interfering RNA targeting GPR137 successfully knocked down GPR137 mRNA and protein expression in both Saos-2 and U2OS cells. In the absence of GPR137, cell viability and colony formation ability were seriously impaired. The extent of apoptosis was also increased in both cell lines. Moreover, AMP-activated protein kinase α, proline-rich AKT substrate of 40 kDa, AKT, and extracellular signal-regulated kinase phosphorylation levels were down-regulated in GPR137 knockdown cells. The results of this study highlight the crucial role of GPR137 in promoting osteosarcoma cell growth in vitro . GPR137 could serve as a potential therapeutic target against osteosarcoma.

Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

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

Teramura, Takeshi, E-mail: teramura@med.kindai.ac.jp; Takehara, Toshiyuki; Onodera, Yuta

2012-01-13

Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells suchmore » as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.« less

Engineered M13 bacteriophage nanocarriers for intracellular delivery of exogenous proteins to human prostate cancer cells.

PubMed

DePorter, Sandra M; McNaughton, Brian R

2014-09-17

The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.

A microfluidics assay to study invasion of human placental trophoblast cells.

PubMed

Abbas, Yassen; Oefner, Carolin Melati; Polacheck, William J; Gardner, Lucy; Farrell, Lydia; Sharkey, Andrew; Kamm, Roger; Moffett, Ashley; Oyen, Michelle L

2017-05-01

Pre-eclampsia, fetal growth restriction and stillbirth are major pregnancy disorders throughout the world. The underlying pathogenesis of these diseases is defective placentation characterized by inadequate invasion of extravillous placental trophoblast cells into the uterine arteries. How trophoblast invasion is controlled remains an unanswered question but is influenced by maternal uterine immune cells called decidual natural killer cells. Here, we describe an in vitro microfluidic invasion assay to study the migration of primary human trophoblast cells. Each experiment can be performed with a small number of cells making it possible to conduct research on human samples despite the challenges of isolating primary trophoblast cells. Cells are exposed to a chemical gradient and tracked in a three-dimensional microenvironment using real-time high-resolution imaging, so that dynamic readouts on cell migration such as directionality, motility and velocity are obtained. The microfluidic system was validated using isolated trophoblast and a gradient of granulocyte-macrophage colony-stimulating factor, a cytokine produced by activated decidual natural killer cells. This microfluidic model provides detailed analysis of the dynamics of trophoblast migration compared to previous assays and can be modified in future to study in vitro how human trophoblast behaves during placentation. © 2017 The Authors.

EZ spheres: a stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs.

PubMed

Ebert, Allison D; Shelley, Brandon C; Hurley, Amanda M; Onorati, Marco; Castiglioni, Valentina; Patitucci, Teresa N; Svendsen, Soshana P; Mattis, Virginia B; McGivern, Jered V; Schwab, Andrew J; Sareen, Dhruv; Kim, Ho Won; Cattaneo, Elena; Svendsen, Clive N

2013-05-01

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology. Copyright © 2013 Elsevier B.V. All rights reserved.

Cellular manganese content is developmentally regulated in human dopaminergic neurons

NASA Astrophysics Data System (ADS)

Kumar, Kevin K.; Lowe, Edward W., Jr.; Aboud, Asad A.; Neely, M. Diana; Redha, Rey; Bauer, Joshua A.; Odak, Mihir; Weaver, C. David; Meiler, Jens; Aschner, Michael; Bowman, Aaron B.

2014-10-01

Manganese (Mn) is both an essential biological cofactor and neurotoxicant. Disruption of Mn biology in the basal ganglia has been implicated in the pathogenesis of neurodegenerative disorders, such as parkinsonism and Huntington's disease. Handling of other essential metals (e.g. iron and zinc) occurs via complex intracellular signaling networks that link metal detection and transport systems. However, beyond several non-selective transporters, little is known about the intracellular processes regulating neuronal Mn homeostasis. We hypothesized that small molecules that modulate intracellular Mn could provide insight into cell-level Mn regulatory mechanisms. We performed a high throughput screen of 40,167 small molecules for modifiers of cellular Mn content in a mouse striatal neuron cell line. Following stringent validation assays and chemical informatics, we obtained a chemical `toolbox' of 41 small molecules with diverse structure-activity relationships that can alter intracellular Mn levels under biologically relevant Mn exposures. We utilized this toolbox to test for differential regulation of Mn handling in human floor-plate lineage dopaminergic neurons, a lineage especially vulnerable to environmental Mn exposure. We report differential Mn accumulation between developmental stages and stage-specific differences in the Mn-altering activity of individual small molecules. This work demonstrates cell-level regulation of Mn content across neuronal differentiation.

Establishment and characterization of a goat synovial membrane cell line susceptible to small ruminant lentivirus infection.

PubMed

Rolland, Morgane; Chauvineau, Cécile; Valas, Stephen; Mamoun, Robert Z; Perrin, Gérard

2004-06-15

Primary goat synovial membrane (GSM) cells are widely used to study small ruminant lentiviruses (SRLV), i.e. maedi visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV), but their limited life-span of 15-20 passages in vitro is problematic. Here, we report that ectopic expression of the catalytic subunit of human telomerase (hTERT) was sufficient to immortalize primary GSM cells. Cultures of hTERT-transfected GSM cells have been passaged for 2 years without showing any phenotypic difference from the original primary GSM cells. The hTERT-transfected cells continued to grow beyond a population doubling number of 250, while no net telomere lengthening was observed for these cells. Moreover, the immortalized GSM cells were susceptible to infection by both CAEV and MVV and were able to propagate theses viruses. Such cell line provides a useful source of standard and robust cells for both research and veterinary purposes.

Cheering for Team Science | Office of Cancer Genomics

Cancer.gov

As a graduate student, my PhD thesis focused on the function of a single human gene, within a genome of some 20,000 genes. Although this sometimes made my work seem insignificant, I was reminded of how important one small piece of a large puzzle can be when I discovered all the ways the gene knockout cells were disadvantaged. Studying the basic biology of our cells made me appreciate the beautiful complexity of human biology.

The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo.

PubMed

Okada, Masashi; Kuramoto, Kenta; Takeda, Hiroyuki; Watarai, Hikaru; Sakaki, Hirotsugu; Seino, Shizuka; Seino, Manabu; Suzuki, Shuhei; Kitanaka, Chifumi

2016-05-10

A phase 2 clinical trial investigating the efficacy and safety of AS602801, a newly developed JNK inhibitor, in the treatment of inflammatory endometriosis is complete. We are now examining whether AS602801 acts against human cancer cells in vitro and in vivo. In vitro, AS602801 exhibited cytotoxicity against both serum-cultured non-stem cancer cells and cancer stem cells derived from human pancreatic cancer, non-small cell lung cancer, ovarian cancer and glioblastoma at concentrations that did not decrease the viability of normal human fibroblasts. AS602801 also inhibited the self-renewal and tumor-initiating capacity of cancer stem cells surviving AS602801 treatment. Cancer stem cells in established xenograft tumors were reduced by systemic administration of AS602801 at a dose and schedule that did not adversely affect the health of the tumor-bearing mice. These findings suggest AS602801 is a promising anti-cancer stem cell agent, and further investigation of the utility of AS602801 in the treatment of cancer seems warranted.

Migratory potential of transplanted glial progenitors as critical factor for successful translation of glia replacement therapy: The gap between mice and men.

PubMed

Srivastava, Rohit K; Bulte, Jeff W M; Walczak, Piotr; Janowski, Miroslaw

2018-05-01

Neurological disorders are a major threat to public health. Stem cell-based regenerative medicine is now a promising experimental paradigm for its treatment, as shown in pre-clinical animal studies. Initial attempts have been on the replacement of neuronal cells only, but glial progenitors (GPs) are now becoming strong alternative cellular therapeutic candidates to replace oligodendrocytes and astrocytes as knowledge accumulates about their important emerging role in various disease processes. There are many examples of successful therapeutic outcomes for transplanted GPs in small animal models, but clinical translation has proved to be challenging due to the 1,000-fold larger volume of the human brain compared to mice. Human GPs transplanted into the mouse brain migrate extensively and can induce global cell replacement, but a similar extent of migration in the human brain would only allow for local rather than global cell replacement. We review here the mechanisms that govern cell migration, which could potentially be exploited to enhance the migratory properties of GPs through cell engineering pre-transplantation. We furthermore discuss the (dis)advantages of the various cell delivery routes that are available, with particular emphasis on intra-arterial injection as the most suitable route for achieving global cell distribution in the larger brain. Now that therapeutic success has proven to be feasible in small animal models, future efforts will need to be directed to enhance global cell delivery and migration to make bench-to-bedside translation a reality. © 2017 Wiley Periodicals, Inc.

Small RNAs Targeting Transcription Start Site Induce Heparanase Silencing through Interference with Transcription Initiation in Human Cancer Cells

PubMed Central

Pu, Jiarui; Mei, Hong; Zhao, Jun; Huang, Kai; Zeng, Fuqing; Tong, Qiangsong

2012-01-01

Heparanase (HPA), an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in human cells. In this study, transfection of siRNA against −9/+10 bp (siH3), but not −174/−155 bp (siH1) or −134/−115 bp (siH2) region relative to transcription start site (TSS) locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 27 trimethylation (H3K27me3) or active chromatin marker acetylated histone H3 (AcH3). The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB), but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (−9/+10 bp) into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells. PMID:22363633

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein.

PubMed

Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro; Ogihara, Takuo

2016-08-05

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. Copyright © 2016 Elsevier Inc. All rights reserved.

Benzoxathiol derivative BOT-4-one suppresses L540 lymphoma cell survival and proliferation via inhibition of JAK3/STAT3 signaling

PubMed Central

Kim, Byung Hak; Min, Yun Sook; Choi, Jung Sook; Baeg, Gyeong-Hun; Kim, Youngsoo; Shin, Jong Wook; Kim, Tae-Yoon

2011-01-01

Persistently activated JAK/STAT3 signaling pathway plays a pivotal role in various human cancers including major carcinomas and hematologic tumors, and is implicated in cancer cell survival and proliferation. Therefore, inhibition of JAK/STAT3 signaling may be a clinical application in cancer therapy. Here, we report that 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo [1,3]oxathiol-4-one (BOT-4-one), a small molecule inhibitor of JAK/STAT3 signaling, induces apoptosis through inhibition of STAT3 activation. BOT-4-one suppressed cytokine (upd)-induced tyrosine phosphorylation and transcriptional activity of STAT92E, the sole Drosophila STAT homolog. Consequently, BOT-4-one significantly inhibited STAT3 tyrosine phosphorylation and expression of STAT3 downstream target gene SOCS3 in various human cancer cell lines, and its effect was more potent in JAK3-activated Hodgkin's lymphoma cell line than in JAK2-activated breast cancer and prostate cancer cell lines. In addition, BOT-4-one-treated Hodgkin's lymphoma cells showed decreased cell survival and proliferation by inducing apoptosis through down-regulation of STAT3 downstream target anti-apoptotic gene expression. These results suggest that BOT-4-one is a novel small molecule inhibitor of JAK3/STAT3 signaling and may have therapeutic potential in the treatment of human cancers harboring aberrant JAK3/STAT3 signaling, specifically Hodgkin's lymphoma. PMID:21499010

Benzoxathiol derivative BOT-4-one suppresses L540 lymphoma cell survival and proliferation via inhibition of JAK3/STAT3 signaling.

PubMed

Kim, Byung Hak; Min, Yun Sook; Choi, Jung Sook; Baeg, Gyeong Hun; Kim, Young Soo; Shin, Jong Wook; Kim, Tae Yoon; Ye, Sang Kyu

2011-05-31

Persistently activated JAK/STAT3 signaling pathway plays a pivotal role in various human cancers including major carcinomas and hematologic tumors, and is implicated in cancer cell survival and proliferation. Therefore, inhibition of JAK/STAT3 signaling may be a clinical application in cancer therapy. Here, we report that 2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo [1,3]oxathiol-4-one (BOT-4-one), a small molecule inhibitor of JAK/STAT3 signaling, induces apoptosis through inhibition of STAT3 activation. BOT-4-one suppressed cytokine (upd)-induced tyrosine phosphorylation and transcriptional activity of STAT92E, the sole Drosophila STAT homolog. Consequently, BOT-4-one significantly inhibited STAT3 tyrosine phosphorylation and expression of STAT3 downstream target gene SOCS3 in various human cancer cell lines, and its effect was more potent in JAK3-activated Hodgkin's lymphoma cell line than in JAK2-activated breast cancer and prostate cancer cell lines. In addition, BOT-4-one-treated Hodgkin's lymphoma cells showed decreased cell survival and proliferation by inducing apoptosis through down-regulation of STAT3 downstream target anti-apoptotic gene expression. These results suggest that BOT-4-one is a novel small molecule inhibitor of JAK3/STAT3 signaling and may have therapeutic potential in the treatment of human cancers harboring aberrant JAK3/STAT3 signaling, specifically Hodgkin's lymphoma.

HEp-2 cell image classification method based on very deep convolutional networks with small datasets

NASA Astrophysics Data System (ADS)

Lu, Mengchi; Gao, Long; Guo, Xifeng; Liu, Qiang; Yin, Jianping

2017-07-01

Human Epithelial-2 (HEp-2) cell images staining patterns classification have been widely used to identify autoimmune diseases by the anti-Nuclear antibodies (ANA) test in the Indirect Immunofluorescence (IIF) protocol. Because manual test is time consuming, subjective and labor intensive, image-based Computer Aided Diagnosis (CAD) systems for HEp-2 cell classification are developing. However, methods proposed recently are mostly manual features extraction with low accuracy. Besides, the scale of available benchmark datasets is small, which does not exactly suitable for using deep learning methods. This issue will influence the accuracy of cell classification directly even after data augmentation. To address these issues, this paper presents a high accuracy automatic HEp-2 cell classification method with small datasets, by utilizing very deep convolutional networks (VGGNet). Specifically, the proposed method consists of three main phases, namely image preprocessing, feature extraction and classification. Moreover, an improved VGGNet is presented to address the challenges of small-scale datasets. Experimental results over two benchmark datasets demonstrate that the proposed method achieves superior performance in terms of accuracy compared with existing methods.

Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling

PubMed Central

Loo, Jacky F.C.; Xia, Dajin; Gao, Sizhi P.; Ma, Zhongjun; Chen, Zhe

2016-01-01

The signal transducers and activators of transcription 3 (STAT3) signaling pathway plays critical roles in the pathogenesis and progression of various human cancers, including non-small cell lung cancer (NSCLC). In this study, we aimed to evaluate the therapeutic potential of physalin A, a bioactive withanolide derived from Physalis alkekengi var. francheti used in traditional Chinese medicine, was evaluated in human NSCLC cells. Its and determined whether it effect oninhibited both constitutive and induced STAT3 activity, through repressing the phosphorylation levels of JAK2 and JAK3, resulting in anti-proliferation and pro-apoptotic effects on NSCLC cells was also determined, and. theThe antitumor effects of physalin A were also validated usingin an in vivo mouse xenograft models of NSCLC cells. Physalin A had anti-proliferative and pro-apoptotic effects in NSCLC cells with constitutively activated STAT3; it also suppressed both constitutive and induced STAT3 activity by modulating the phosphorylation of JAK2 and JAK3. Furthermore, physalin A abrogated the nuclear translocation and transcriptional activity of STAT3, thereby decreasing the expression levels of STAT3, its target genes, such as Bcl-2 and XIAP. Knockdown of STAT3 expression by small interfering RNA (siRNA) significantly enhanced the pro-apoptotic effects of physalin A in NSCLC cells. Moreover, physalin A significantly suppressed tumor xenograft growth. Thus, as an inhibitor of JAK2/3-STAT3 signaling, physalin A, has potent anti-tumor activities, which may facilitate the development of a therapeutic strategy for treating NSCLC. PMID:26843613

Targeting 6-phosphogluconate dehydrogenase in the oxidative PPP sensitizes leukemia cells to antimalarial agent dihydroartemisinin.

PubMed

Elf, S; Lin, R; Xia, S; Pan, Y; Shan, C; Wu, S; Lonial, S; Gaddh, M; Arellano, M L; Khoury, H J; Khuri, F R; Lee, B H; Boggon, T J; Fan, J; Chen, J

2017-01-12

The oxidative pentose phosphate pathway (PPP) is crucial for cancer cell metabolism and tumor growth. We recently reported that targeting a key oxidative PPP enzyme, 6-phosphogluconate dehydrogenase (6PGD), using our novel small-molecule 6PGD inhibitors Physcion and its derivative S3, shows anticancer effects. Notably, humans with genetic deficiency of either 6PGD or another oxidative PPP enzyme, glucose-6-phosphate dehydrogenase, exhibit non-immune hemolytic anemia upon exposure to aspirin and various antimalarial drugs. Inspired by these clinical observations, we examined the anticancer potential of combined treatment with 6PGD inhibitors and antimalarial drugs. We found that stable knockdown of 6PGD sensitizes leukemia cells to antimalarial agent dihydroartemisinin (DHA). Combined treatment with DHA and Physcion activates AMP-activated protein kinase, leading to synergistic inhibition of human leukemia cell viability. Moreover, our combined therapy synergistically attenuates tumor growth in xenograft nude mice injected with human K562 leukemia cells and cell viability of primary leukemia cells from human patients, but shows minimal toxicity to normal hematopoietic cells in mice as well as red blood cells and mononucleocytes from healthy human donors. Our findings reveal the potential for combined therapy using optimized doses of Physcion and DHA as a novel antileukemia treatment without inducing hemolysis.

Identification of novel kynurenine production-inhibiting benzenesulfonamide derivatives in cancer cells.

PubMed

Nakano, Shintaro; Takai, Kazushige; Isaka, Yoshinobu; Takahashi, Susumu; Unno, Yuka; Ogo, Naohisa; Matsuno, Kenji; Takikawa, Osamu; Asai, Akira

2012-03-16

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is known to be a key regulator of human immune responses including cancer immune tolerance. Therefore, abrogation of Kyn production from cancer cells by small molecules may be a promising approach to anticancer therapy. Indeed, several small molecule inhibitors of indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the catabolism of Trp to Kyn, exert antitumor effects in animal models. We screened our chemical libraries using a cell-based Kyn production assay to identify a new type of small molecules that regulate Kyn production, and for the first time identified a benzenesulfonamide derivative (compound 1) as a hit with the ability to inhibit Kyn production in interferon-γ (IFN-γ)-stimulated A431 and HeLa cells. Unlike the previously identified S-benzylisothiourea derivative, compound 2, compound 1 had little effect on the enzymatic activity of recombinant human IDO in vitro but suppressed the expression of IDO at the mRNA level in cells. Furthermore, compound 1 suppressed STAT1-dependent transcriptional activity and DNA binding, whereas no decrement in either the expression or phosphorylation level of STAT1 was observed. The inhibition of IDO expression by several benzenesulfonamide derivatives is associated with the suppression of STAT1. Thus, compound 1 and its analogs might be useful for analyzing the regulation of IDO activation, and STAT1-targeting could be an alternative to the IDO-directed approach for the regulation of Kyn levels by small molecules in the tumor microenvironment. Copyright © 2012 Elsevier Inc. All rights reserved.

Human embryonic stem cells express a unique set of microRNAs.

PubMed

Suh, Mi-Ra; Lee, Yoontae; Kim, Jung Yeon; Kim, Soo-Kyoung; Moon, Sung-Hwan; Lee, Ji Yeon; Cha, Kwang-Yul; Chung, Hyung Min; Yoon, Hyun Soo; Moon, Shin Yong; Kim, V Narry; Kim, Kye-Seong

2004-06-15

Human embryonic stem (hES) cells are pluripotent cell lines established from the explanted inner cell mass of human blastocysts. Despite their importance for human embryology and regenerative medicine, studies on hES cells, unlike those on mouse ES (mES) cells, have been hampered by difficulties in culture and by scant knowledge concerning the regulatory mechanism. Recent evidence from plants and animals indicates small RNAs of approximately 22 nucleotides (nt), collectively named microRNAs, play important roles in developmental regulation. Here we describe 36 miRNAs (from 32 stem-loops) identified by cDNA cloning in hES cells. Importantly, most of the newly cloned miRNAs are specifically expressed in hES cells and downregulated during development into embryoid bodies (EBs), while miRNAs previously reported from other human cell types are poorly expressed in hES cells. We further show that some of the ES-specific miRNA genes are highly related to each other, organized as clusters, and transcribed as polycistronic primary transcripts. These miRNA gene families have murine homologues that have similar genomic organizations and expression patterns, suggesting that they may operate key regulatory networks conserved in mammalian pluripotent stem cells. The newly identified hES-specific miRNAs may also serve as molecular markers for the early embryonic stage and for undifferentiated hES cells.

Deletion of Cytoplasmic Double-Stranded RNA Sensors Does Not Uncover Viral Small Interfering RNA Production in Human Cells.

PubMed

Schuster, Susan; Tholen, Lotte E; Overheul, Gijs J; van Kuppeveld, Frank J M; van Rij, Ronald P

2017-01-01

Antiviral immunity in insects and plants is mediated by the RNA interference (RNAi) pathway in which viral long double-stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer enzymes. Although this pathway is evolutionarily conserved, its involvement in antiviral defense in mammals is the subject of debate. In vertebrates, recognition of viral RNA induces a sophisticated type I interferon (IFN)-based immune response, and it has been proposed that this response masks or inhibits antiviral RNAi. To test this hypothesis, we analyzed viral small RNA production in differentiated cells deficient in the cytoplasmic RNA sensors RIG-I and MDA5. We did not detect 22-nucleotide (nt) viral siRNAs upon infection with three different positive-sense RNA viruses. Our data suggest that the depletion of cytoplasmic RIG-I-like sensors is not sufficient to uncover viral siRNAs in differentiated cells. IMPORTANCE The contribution of the RNA interference (RNAi) pathway in antiviral immunity in vertebrates has been widely debated. It has been proposed that RNAi possesses antiviral activity in mammalian systems but that its antiviral effect is masked by the potent antiviral interferon response in differentiated mammalian cells. In this study, we show that inactivation of the interferon response is not sufficient to uncover antiviral activity of RNAi in human epithelial cells infected with three wild-type positive-sense RNA viruses.

Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model.

PubMed

Koskimaki, Jacob E; Karagiannis, Emmanouil D; Tang, Benjamin C; Hammers, Hans; Watkins, D Neil; Pili, Roberto; Popel, Aleksander S

2010-02-01

Angiogenesis is the formation of neovasculature from a pre-existing vascular network. Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions in vitro in human umbilical vein endothelial cell (HUVEC) proliferation and migration assays. One family of peptides with high activity is derived from the alpha-fibrils of type IV collagen. Based on the results from the in vitro screening, we have evaluated the ability of a 20 amino acid peptide derived from the alpha5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed in vivo angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line. Pentastatin-1 decreased the invasion of vessels into angioreactors in vivo in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density in vivo in a small cell lung cancer xenograft model. The peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer.

A Novel Proteomics Approach to Identify SUMOylated Proteins and Their Modification Sites in Human Cells*

PubMed Central

Galisson, Frederic; Mahrouche, Louiza; Courcelles, Mathieu; Bonneil, Eric; Meloche, Sylvain; Chelbi-Alix, Mounira K.; Thibault, Pierre

2011-01-01

The small ubiquitin-related modifier (SUMO) is a small group of proteins that are reversibly attached to protein substrates to modify their functions. The large scale identification of protein SUMOylation and their modification sites in mammalian cells represents a significant challenge because of the relatively small number of in vivo substrates and the dynamic nature of this modification. We report here a novel proteomics approach to selectively enrich and identify SUMO conjugates from human cells. We stably expressed different SUMO paralogs in HEK293 cells, each containing a His6 tag and a strategically located tryptic cleavage site at the C terminus to facilitate the recovery and identification of SUMOylated peptides by affinity enrichment and mass spectrometry. Tryptic peptides with short SUMO remnants offer significant advantages in large scale SUMOylome experiments including the generation of paralog-specific fragment ions following CID and ETD activation, and the identification of modified peptides using conventional database search engines such as Mascot. We identified 205 unique protein substrates together with 17 precise SUMOylation sites present in 12 SUMO protein conjugates including three new sites (Lys-380, Lys-400, and Lys-497) on the protein promyelocytic leukemia. Label-free quantitative proteomics analyses on purified nuclear extracts from untreated and arsenic trioxide-treated cells revealed that all identified SUMOylated sites of promyelocytic leukemia were differentially SUMOylated upon stimulation. PMID:21098080

Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells.

PubMed

Korytnikov, Roman; Nostro, Maria Cristina

2016-05-15

Generation of pancreatic β-cells from human pluripotent stem cells (hPSCs) has enormous importance in type 1 diabetes (T1D), as it is fundamental to a treatment strategy based on cellular therapeutics. Being able to generate β-cells, as well as other mature pancreatic cells, from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) will also enable the development of platforms that can be used for disease modeling and drug testing for a variety of pancreas-associated diseases, including cystic fibrosis. For this to occur, it is crucial to develop differentiation strategies that are robust and reproducible across cell lines and laboratories. In this article we describe two serum-free differentiation protocols designed to generate specific pancreatic lineages from hPSCs. Our approach employs a variety of cytokines and small molecules to mimic developmental pathways active during pancreatic organogenesis and allows for the in vitro generation of distinct pancreatic populations. The first protocol is designed to give rise to polyhormonal cells that have the potential to differentiate into glucagon-producing cells. The second protocol is geared to generate multipotent pancreatic progenitor cells, which harbor the potential to generate all pancreatic lineages including: monohormonal endocrine cells, acinar, and ductal cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Pembrolizumab combined with stereotactic body radiotherapy in a patient with human immunodeficiency virus and advanced non-small cell lung cancer: a case report.

PubMed

Li, Dongqi; He, Chuanchun; Xia, Yaoxiong; Du, Yaxi; Zhang, Jing

2018-04-23

Pembrolizumab has significantly improved outcomes in patients with advanced non-small cell lung cancer. Combining programmed death-1 inhibitor with stereotactic body radiotherapy showed a slight toxicity and good benefits in recent clinical trials. However, patients infected with human immunodeficiency virus were excluded from most trials because it was assumed that their anti-tumor immunity was compromised compared with immunocompetent patients. In June 2016, a 52-year-old Chinese man presented with human immunodeficiency virus and lung adenocarcinoma (T1bN3M1b). From November 2016 to December 2016, systemic chemotherapy and palliative radiotherapy for bone metastasis of femoral neck were carried out, but the tumor progressed. In January 2017, after immunochemistry detection of programmed death-1 and programmed death-ligand 1 expression (both > 50%), pembrolizumab was started. Three weeks after pembrolizumab, we combined stereotactic body radiotherapy for the primary lung tumor. He received no comfort and his CD4 lymphocyte count was stable. Human immunodeficiency virus-ribonucleic acid remained below the limits of detection. In March 2017, after three cycles of pembrolizumab and 5 weeks of stereotactic body radiotherapy therapy, he suddenly presented with palpitations. Emergency computed tomography scanning showed massive pericardial effusion and interstitial pneumonia. So we interrupted the pembrolizumab use and initiated treatment with prednisolone 1 mg/kg; however, the tumor progressed. Then, his CD4 lymphocyte count declined. Finally he died in June 2017 due to dyscrasia. Pembrolizumab combined with SBRT therapy for patients with human immunodeficiency virus infection and non-small cell lung cancer may lead to serious immune-related adverse events and more clinical trials are needed.

Simultaneous Multiparameter Cellular Energy Metabolism Profiling of Small Populations of Cells.

PubMed

Kelbauskas, Laimonas; Ashili, Shashaanka P; Lee, Kristen B; Zhu, Haixin; Tian, Yanqing; Meldrum, Deirdre R

2018-03-12

Functional and genomic heterogeneity of individual cells are central players in a broad spectrum of normal and disease states. Our knowledge about the role of cellular heterogeneity in tissue and organism function remains limited due to analytical challenges one encounters when performing single cell studies in the context of cell-cell interactions. Information based on bulk samples represents ensemble averages over populations of cells, while data generated from isolated single cells do not account for intercellular interactions. We describe a new technology and demonstrate two important advantages over existing technologies: first, it enables multiparameter energy metabolism profiling of small cell populations (<100 cells)-a sample size that is at least an order of magnitude smaller than other, commercially available technologies; second, it can perform simultaneous real-time measurements of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and mitochondrial membrane potential (MMP)-a capability not offered by any other commercially available technology. Our results revealed substantial diversity in response kinetics of the three analytes in dysplastic human epithelial esophageal cells and suggest the existence of varying cellular energy metabolism profiles and their kinetics among small populations of cells. The technology represents a powerful analytical tool for multiparameter studies of cellular function.

Chemical Carcinogen-Induced Changes in tRNA Metabolism in Human Cells

DTIC Science & Technology

1982-11-01

R.W.T., M.S.E.) Department of Medical Microbiology & Immunology (R.G.) Department of Medicine (M.R.G.) The Ohio State University Columbus, Ohio...1973): The measurement of radioactive precursor incorporation into small monolayer cultures. In: Methods In Cell Bioloqy, edited by D.M. Prescott , Vol

Reptin drives tumour progression and resistance to chemotherapy in non-small cell lung cancer.

PubMed

Mikesch, Jan-Henrik; Schwammbach, Daniela; Hartmann, Wolfgang; Schmidt, Lars H; Schliemann, Christoph; Angenendt, Linus; Wiewrodt, Rainer; Marra, Alessandro; Thoennissen, Nils H; Wardelmann, Eva; Köhler, Gabriele; Lenz, Georg; Müller-Tidow, Carsten; Berdel, Wolfgang E; Arteaga, Maria-Francisca

2018-05-31

While targeted non-small cell lung cancer (NSCLC) therapies improved outcome of defined disease subtypes, prognosis of most of the patients remains poor. We found the AAA+ ATPase Reptin to be highly expressed in the vast majority of 278 NSCLC tumour samples. Thus, the objective of the study was to assess the role of Reptin in NSCLC.Survival analyses of 1,145 NSCLC patients revealed that high RNA expression levels of Reptin are associated with adverse outcome. Knock down of Reptin in human NSCLC cells impaired growth ex vivo and eliminated engraftment in a xenograft model. We uncovered direct interaction of Reptin with histone deacetylase 1 (HDAC1), as the critical mechanism driving NSCLC tumour progression. Pharmacological disruption of Reptin/HDAC1 complex resulted in substantial decrease of NSCLC cell proliferation and induced significant sensitization to cisplatin.In conclusion, our results identify Reptin as a novel independent prognostic factor and as a key regulator mediating proliferation and clonal growth of human NSCLC cells ex vivo and in vivo We unveil a Reptin/HDAC1 protein complex whose pharmacological disruption sensitizes NSCLC cells to cisplatin, suggesting this approach for application in clinical trials. Copyright ©ERS 2018.

The therapeutic response of CDDO-Me in the esophageal squamous cell carcinoma (ESCC) cells is mediated by CaMKIIα.

PubMed

Wang, Yan-Yang; Zhou, Shun; Zhao, Ren; Hai, Ping; Zhe, Hong

2016-01-01

CDDO-Me has exhibited a potent anticancer effect in human esophageal squamous cell carcinoma (ESCC) cells in our previous study, but the molecular interactome remains elusive. We applied the approach of stable-isotope labeling by amino acids in cell culture (SILAC) to assess the proteomic responses of CDDO-Me treatment in human ESCC Ec109 cells. The data were subsequently validated using Western blot assay. The results of our study revealed that CDDO-Me increased the expression level of 543 protein molecules, but decreased the expression level of 709 protein molecules in Ec109 cells. Among these modulated protein molecules, calcium/calmodulin-dependent protein kinase type II subunit α (CaMKIIα) was highly expressed in all tested ESCC cell lines, whereas its expression levels were substantially lower in normal control cell line. Its silencing by small interfering RNA inhibited CDDO-Me induced apoptosis and autophagy in ESCC cells. Collectively, these data demonstrate that the therapeutic response of CDDO-Me in the human ESCC cells is mediated by CaMKIIα.

Oxidative stress in Nipah virus-infected human small airway epithelial cells.

PubMed

Escaffre, Olivier; Halliday, Hailey; Borisevich, Viktoriya; Casola, Antonella; Rockx, Barry

2015-10-01

Nipah virus (NiV) is a zoonotic emerging pathogen that can cause severe and often fatal respiratory disease in humans. The pathogenesis of NiV infection of the human respiratory tract remains unknown. Reactive oxygen species (ROS) produced by airway epithelial cells in response to viral infections contribute to lung injury by inducing inflammation and oxidative stress; however, the role of ROS in NiV-induced respiratory disease is unknown. To investigate whether NiV induces oxidative stress in human respiratory epithelial cells, we used oxidative stress markers and monitored antioxidant gene expression. We also used ROS scavengers to assess their role in immune response modulation. Oxidative stress was confirmed in infected cells and correlated with the reduction in antioxidant enzyme gene expression. Infected cells treated by ROS scavengers resulted in a significant decrease of the (F2)-8-isoprostane marker, inflammatory responses and virus replication. In conclusion, ROS are induced during NiV infection in human respiratory epithelium and contribute to the inflammatory response. Understanding how oxidative stress contributes to NiV pathogenesis is crucial for therapeutic development.

[Neuronal organization of thalamic nucleus reticularis in adult man].

PubMed

Berezhnaia, L A

2005-01-01

The neuronal content of human thalamic nucleus reticularis was studied in serial sections cut in sagittal and frontal projections and impregnated with silver nitrate using Golgi method. The neuronal content of human thalamic nucleus reticularis was found to be more diverse than previously reported in animals and man. Besides two types of sparsely-branched long-dendritic spineless R1 and R2 neurons, this nucleus contained spiny cells. Medium and small-sized sparsely-branched short-dendritic neurons and densely-branched spiny cells were demonstrated. The principle of organization of human thalamic nucleus reticularis is described.

Evaluation of EML4-ALK Fusion Proteins in Non-Small Cell Lung Cancer Using Small Molecule Inhibitors12

PubMed Central

Li, Yongjun; Ye, Xiaofen; Liu, Jinfeng; Zha, Jiping; Pei, Lin

2011-01-01

The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non-small cell lung cancer and is mutually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non-small cell lung cancer (NSCLC). We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mechanism of EML4-ALK inhibition by a small molecule inhibitor. PMID:21245935

The investigation of ceranib-2 on apoptosis and drug interaction with carboplatin in human non small cell lung cancer cells in vitro.

PubMed

Yildiz-Ozer, Merve; Oztopcu-Vatan, Pinar; Kus, Gokhan

2018-02-01

Ceramide is found to be involved in inhibition of cell division and induction of apoptosis in certain tumour cells. Ceranib-2 is an agent that increases ceramide levels by inhibiting ceramidase in cancer cells. Therefore, we aimed to investigate the effects of ceranib-2 on cell survival, apoptosis and interaction with carboplatin in human non-small cell lung cancer cells. The cytotoxic effect of ceranib-2 (1-100 µM) was determined by MTT assay in human lung adenocarcinoma (A549) and large cell lung carcinoma (H460) cells. Carboplatin (1-100 µM) and lung bronchial epithelial cells (BEAS-2B) were used as positive controls. Morphological and ultrastructural changes were analysed by light microscope and TEM. Apoptotic/necrotic cell death and acid ceramidase activity were analysed by ELISA. Combination effects of ceranib-2 and carboplatin were investigated by MTT. The expression levels of CASP3, CASP9, BAX and BCL-2 were examined by qRT-PCR. The IC 50 of ceranib-2 was determined as 22 μM in A549 cells and 8 μM in H460 cells for 24 h. Morphological changes and induction of DNA fragmentation have revealed apoptotic effects of ceranib-2 in both cell lines. Ceranib-2 and carboplatin has shown synergism in combined treatment at 10 and 25 μM doses in H460 cells for 24 h. Ceranib-2 inhibited acid ceramidase activity by 44% at 25 µM in H460 cells. Finally, CASP3, CASP9 and BAX expressions were increased while BCL-2 expression was reduced in both cells. Our results obtained some preliminary results about the cytotoxic and apoptotic effects of ceranib-2 for the first time in NSCLC cell lines.

Establishment of conditionally immortalized human glomerular mesangial cells in culture, with unique migratory properties.

PubMed

Sarrab, Ramadan M; Lennon, Rachel; Ni, Lan; Wherlock, Matthew D; Welsh, Gavin I; Saleem, Moin A

2011-11-01

The aim of this study was to establish an immortalized human mesangial cell line similar to mesangial cells in vivo for use as a tool for understanding glomerular cell function. Mesangial cells were isolated from glomerular outgrowths from a normal human kidney, then retrovirally transfected with a temperature-sensitive SV40T antigen+human telomerase (hTERT). Mesangial cells exhibited features of compact cells with small bodies in a confluent monolayer at 33°C, but the cell shape changed to flat and stellate after 5 days in growth-restrictive conditions (37°C). Western blot and immunofluorescence analysis showed that podocyte markers (nephrin, CD2AP, podocin, Wilms' tumor-1) and an endothelial-specific molecule (VE-cadherin) were not detectable in this cell line, whereas markers characteristic of mesangial cells (α-SMA, fibronectin, and PDGFβ-R) were strongly expressed. In migration assays, a significant reduction in wound surface was observed in podocyte and endothelial cells as soon as 12 h (75 and 62%, respectively) and complete wound closure after 24 h. In contrast, no significant change was observed in mesangial cells after 12 h, and even after 48 h the wounds were not completely closed. Until now, conditionally immortalized podocyte and endothelial cell lines derived from mice and humans have been described, and this has greatly boosted research on glomerular physiology and pathology. We have established the first conditionally immortalized human glomerular mesangial cell line, which will be an important adjunct in studies of representative glomerular cells, as well as in coculture studies. Unexpectedly, mesangial cells' ability to migrate seems to be slower than for other glomerular cells, suggesting this line will demonstrate functional properties distinct from previously available mesangial cell cultures. This conditionally immortalized human mesangial cell line represents a new tool for the study of human mesangial cell biology in vitro.

Multiple cis-acting sequence elements are required for efficient splicing of simian virus 40 small-t antigen pre-mRNA.

PubMed Central

Fu, X Y; Colgan, J D; Manley, J L

1988-01-01

We have determined the effects of a number of mutations in the small-t antigen mRNA intron on the alternative splicing pattern of the simian virus 40 early transcript. Expansion of the distance separating the small-t pre-mRNA lariat branch point and the shared large T-small t 3' splice site from 18 to 29 nucleotides (nt) resulted in a relative enhancement of small-t splicing in vivo. This finding, coupled with the observation that large-T pre-RNA splicing in vitro was not affected by this expansion, suggests that small-t splicing is specifically constrained by a short branch point-3' splice site distance. Similarly, the distance separating the 5' splice site and branch point (48 nt) was found to be at or near a minimum for small-t splicing, because deletions in this region as small as 2 nt dramatically reduced the ratio of small-t to large-T mRNA that accumulated in transfected cells. Finally, a specific sequence within the small-t intron, encompassing the upstream branch sites used in large-T splicing, was found to be an important element in the cell-specific pattern of early alternative splicing. Substitutions within this region reduced the ratio of small-t to large-T mRNA produced in HeLa cells but had only minor effects in human 293 cells. Images PMID:2851720

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

PubMed Central

Cabada, Miguel M.; Nichols, Joan; Gomez, Guillermo; White, A. Clinton

2013-01-01

The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens. PMID:23509153

Killing multiple myeloma cells with the small molecule 3-bromopyruvate: implications for therapy.

PubMed

Majkowska-Skrobek, Grażyna; Augustyniak, Daria; Lis, Paweł; Bartkowiak, Anna; Gonchar, Mykhailo; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

2014-07-01

The small molecule 3-bromopyruvate (3-BP), which has emerged recently as the first member of a new class of potent anticancer agents, was tested for its capacity to kill multiple myeloma (MM) cancer cells. Human MM cells (RPMI 8226) begin to lose viability significantly within 8 h of incubation in the presence of 3-BP. The Km (0.3 mmol/l) for intracellular accumulation of 3-BP in MM cells is 24 times lower than that in control cells (7.2 mmol/l). Therefore, the uptake of 3-BP by MM cells is significantly higher than that by peripheral blood mononuclear cells. Further, the IC50 values for human MM cells and control peripheral blood mononuclear cells are 24 and 58 µmol/l, respectively. Therefore, specificity and selectivity of 3-BP toward MM cancer cells are evident on the basis of the above. In MM cells the transcription levels of the gene encoding the monocarboxylate transporter MCT1 is significantly amplified compared with control cells. The level of intracellular ATP in MM cells decreases by over 90% within 1 h after addition of 100 µmol/l 3-BP. The cytotoxicity of 3-BP, exemplified by a marked decrease in viability of MM cells, is potentiated by the inhibitor of glutathione synthesis buthionine sulfoximine. In addition, the lack of mutagenicity and its superior capacity relative to Glivec to kill MM cancer cells are presented in this study.

Depletion of Securin Induces Senescence After Irradiation and Enhances Radiosensitivity in Human Cancer Cells Regardless of Functional p53 Expression

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

Chen Wenshu; Yu Yichu; Lee Yijang

2010-06-01

Purpose: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. Methods and Materials: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, gamma-H2AX, and phospho-Chk2. Senescent cells were analyzed using a beta-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin genemore » knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. Results: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced gamma-H2AX and Chk2 phosphorylation were induced transiently in securin-wild-type cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiation-induced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. Conclusions: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation.« less

Capsaicin Displays Anti-Proliferative Activity against Human Small Cell Lung Cancer in Cell Culture and Nude Mice Models via the E2F Pathway

PubMed Central

Hardman, W. Elaine; Luo, Haitao; Chen, Yi C.; Carpenter, A. Betts; Lau, Jamie K.; Dasgupta, Piyali

2010-01-01

Background Small cell lung cancer (SCLC) is characterized by rapid progression and low survival rates. Therefore, novel therapeutic agents are urgently needed for this disease. Capsaicin, the active ingredient of chilli peppers, displays anti-proliferative activity in prostate and epidermoid cancer in vitro. However, the anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this void of knowledge and explores the anti-proliferative effect of capsaicin in SCLC in vitro and in vivo. Methodology/Principal Findings BrdU assays and PCNA ELISAs showed that capsaicin displays robust anti-proliferative activity in four human SCLC cell lines. Furthermore, capsaicin potently suppressed the growth of H69 human SCLC tumors in vivo as ascertained by CAM assays and nude mice models. The second part of our study attempted to provide insight into molecular mechanisms underlying the anti-proliferative activity of capsaicin. We found that the anti-proliferative activity of capsaicin is correlated with a decrease in the expression of E2F-responsive proliferative genes like cyclin E, thymidylate synthase, cdc25A and cdc6, both at mRNA and protein levels. The transcription factor E2F4 mediated the anti-proliferative activity of capsaicin. Ablation of E2F4 levels by siRNA methodology suppressed capsaicin-induced G1 arrest. ChIP assays demonstrated that capsaicin caused the recruitment of E2F4 and p130 on E2F-responsive proliferative promoters, thereby inhibiting cell proliferation. Conclusions/Significance Our findings suggest that the anti-proliferative effects of capsaicin could be useful in the therapy of human SCLCs. PMID:20421925

Zinc finger X-chromosomal protein (ZFX) promotes solid agar colony growth of osteosarcoma cells.

PubMed

Jiang, Rui; Wang, Jin-cheng; Sun, Mei; Zhang, Xing-yi; Wu, Han

2012-01-01

Zinc finger X-chromosomal protein (ZFX) is a member of the zinc finger family of proteins. The importance of ZFX in several cancer types, including prostate cancer, laryngeal squamous cell carcinoma, and glioma, has been addressed. However, the role of ZFX in human osteosarcoma remains unknown. Here we investigated the phenotype of ZFX knockdown on cell proliferation and in vitro tumorigenesis using lentivirus-mediated loss-of-function strategy. The results demonstrated that the proliferation and colony formation ability of human osteosarcoma Saos-2 and MG63 cells was impaired by ZFX small interfering RNA (siRNA)-expressing lentivirus. Moreover, loss of ZFX led to G0/G1 phase cell cycle arrest and a significant increase of cells in the sub-G1 fraction, indicating that ZFX functions as an oncogene in the malignant proliferation process in osteosarcoma. Furthermore, ZFX siRNA may have an antitumorigenic effect on osteosarcoma cells. Our findings hold important significance for RNA interference-mediated cancer gene therapy for human osteosarcoma.

Temporal morphologic changes in human colorectal carcinomas following xenografting.

PubMed

Barkla, D H; Tutton, P J

1983-03-01

The temporal morphologic changes of human colorectal carcinomas following xenografting into immunosuppressed mice were investigated by the use of light and transmission electron microscopy. The results show that colorectal carcinomas undergo a series of morphologic changes during the initial 30-day period following transplantation. During the initial 1-5-day period the majority of tumor cells die, and during the following 5-10-day period the necrotic debris created during the 1-5-day period is removed by host-supplied inflammatory cells. Only small groups of peripherally placed tumor cells survived at the end of the first 10 days. During the 10-20-day period the tumor cell populations of xenografts were reestablished by a morphologically heterogeneous population of tumor cells, and during the 20-30 day period consolidation of this process continued and some xenografts showed macroscopic evidence of growth. The authors hypothesize that human colorectal carcinomas, like the antecedent epithelium, contain subpopulations of undifferentiated cells that give rise to populations of more-differentiated cells.

Association of Plasmodium falciparum with Human Endothelial Cells in vitro

PubMed Central

Utter, Christopher; Serrano, Adelfa E.; Glod, John W.; Leibowitz, Michael J.

2017-01-01

Endothelial abnormalities play a critical role in the pathogenesis of malaria caused by the human pathogen, Plasmodium falciparum. In serious infections and especially in cerebral malaria, red blood cells infected with the parasite are sequestered in small venules in various organs, resulting in endothelial activation and vascular occlusion, which are believed to be largely responsible for the morbidity and mortality caused by this infection, especially in children. We demonstrate that after incubation with infected red blood cells (iRBCs), cultured human umbilical vein endothelial cells (HUVECs) contain parasite protein, genomic DNA, and RNA, as well as intracellular vacuoles with apparent parasite-derived material, but not engulfed or adherent iRBCs. The association of this material with the HUVECs is observed over 96 hours after removal of iRBCs. This phenomenon may occur in endothelial cells in vivo by the process of trogocytosis, in which transfer of material between cells depends on direct cell contact. This process may contribute to the endothelial activation and disruption involved in the pathogenesis of cerebral malaria. PMID:28656007

The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells

PubMed Central

Munoz, Javier; Low, Teck Y; Kok, Yee J; Chin, Angela; Frese, Christian K; Ding, Vanessa; Choo, Andre; Heck, Albert J R

2011-01-01

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. PMID:22108792

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells.

PubMed

Sluch, Valentin M; Chamling, Xitiz; Liu, Melissa M; Berlinicke, Cynthia A; Cheng, Jie; Mitchell, Katherine L; Welsbie, Derek S; Zack, Donald J

2017-11-01

Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972-1986. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Anti-proliferative and anti-angiogenic effects of CB2R agonist (JWH-133) in non-small lung cancer cells (A549) and human umbilical vein endothelial cells: an in vitro investigation.

PubMed

Vidinský, B; Gál, P; Pilátová, M; Vidová, Z; Solár, P; Varinská, L; Ivanová, L; Mojžíš, J

2012-01-01

Non-small cell lung cancer has one of the highest mortality rates among cancer-suffering patients. It is well known that the unwanted psychotropic effects of cannabinoids (CBs) are mediated via the CB(1) receptor (R), and selective targeting of the CB(2)R would thus avoid side effects in cancer treatment. Therefore, the aim of our study was to evaluate the effect of selective CB(2)R agonist, JWH-133, on A549 cells (non-small lung cancer) and human umbilical vein endothelial cells (HUVECs). Cytotoxicity assay and DNA fragmentation assay were employed to evaluate the influence of JWH-133 (3-(1,1-dimethylbutyl)- 1-deoxy-Δ8-tetrahydrocannabinol) on investigated cancer cells. In addition, migration assay and gelatinase zymography were performed in HUVECs to asses JWH-133 anti-angiogenic activity. Our study showed that JWH-133 exerted cytotoxic effect only at the highest concentration used (10(-4) mol/l), while inhibition of colony formation was also detected at the non-toxic concentrations (10(-5)-10(-8) mol/l). JWH-133 was also found to be able to induce weak DNA fragmentation in A549 cells. Furthermore, JWH-133 at non-toxic concentrations inhibited some steps in the process of angiogenesis. It significantly inhibited endothelial cell migration after 17 h of incubation at concentrations of 10(-4)-10(-6) mol/l. In addition, JWH-133 inhibited MMP-2 secretion as assessed by gelatinase zymography. The present study demonstrates the in vitro anti-proliferative and anti-angiogenic potential of CB(2)R agonist, JWH-133, in nonsmall lung cancer cells and HUVECs. Our results generate a rationale for further in vivo efficacy studies with this compound in preclinical cancer models.

The Nitric Oxide Prodrug JS-K Is Effective against Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo: Involvement of Reactive Oxygen SpeciesS⃞

PubMed Central

Chakrapani, Harinath; Saavedra, Joseph E.; Morris, Nicole L.; Holland, Ryan J.; Kosak, Ken M.; Shami, Paul J.; Anderson, Lucy M.; Keefer, Larry K.

2011-01-01

Non–small-cell lung cancer is among the most common and deadly forms of human malignancies. Early detection is unusual, and there are no curative therapies in most cases. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Here, we show that O2-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a potent cytotoxic agent against a subset of human non–small-cell lung cancer cell lines both in vitro and as xenografts in mice. JS-K treatment led to 75% reduction in the growth of H1703 lung adenocarcinoma cells in vivo. Differences in sensitivity to JS-K in different lung cancer cell lines seem to be related to their endogenous levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS). Other related factors, levels of peroxiredoxin 1 (PRX1) and 8-oxo-deoxyguanosine glycosylase (OGG1), also correlated with drug sensitivity. Treatment of the lung adenocarcinoma cells with JS-K resulted in oxidative/nitrosative stress in cells with high basal levels of ROS/RNS, which, combined with the arylating properties of the compound, was reflected in glutathione depletion and alteration in cellular redox potential, mitochondrial membrane permeabilization, and cytochrome c release. Inactivation of manganese superoxide dismutase by nitration was associated with increased superoxide and significant DNA damage. Apoptosis followed these events. Taken together, the data suggest that diazeniumdiolate-based NO-releasing prodrugs may have application as a personalized therapy for lung cancers characterized by high levels of ROS/RNS. PRX1 and OGG1 proteins, which can be easily measured, could function as biomarkers for identifying tumors sensitive to the therapy. PMID:20962031

The nitric oxide prodrug JS-K is effective against non-small-cell lung cancer cells in vitro and in vivo: involvement of reactive oxygen species.

PubMed

Maciag, Anna E; Chakrapani, Harinath; Saavedra, Joseph E; Morris, Nicole L; Holland, Ryan J; Kosak, Ken M; Shami, Paul J; Anderson, Lucy M; Keefer, Larry K

2011-02-01

Non-small-cell lung cancer is among the most common and deadly forms of human malignancies. Early detection is unusual, and there are no curative therapies in most cases. Diazeniumdiolate-based nitric oxide (NO)-releasing prodrugs are a growing class of promising NO-based therapeutics. Here, we show that O(2)-(2,4-dinitrophenyl)-1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a potent cytotoxic agent against a subset of human non-small-cell lung cancer cell lines both in vitro and as xenografts in mice. JS-K treatment led to 75% reduction in the growth of H1703 lung adenocarcinoma cells in vivo. Differences in sensitivity to JS-K in different lung cancer cell lines seem to be related to their endogenous levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS). Other related factors, levels of peroxiredoxin 1 (PRX1) and 8-oxo-deoxyguanosine glycosylase (OGG1), also correlated with drug sensitivity. Treatment of the lung adenocarcinoma cells with JS-K resulted in oxidative/nitrosative stress in cells with high basal levels of ROS/RNS, which, combined with the arylating properties of the compound, was reflected in glutathione depletion and alteration in cellular redox potential, mitochondrial membrane permeabilization, and cytochrome c release. Inactivation of manganese superoxide dismutase by nitration was associated with increased superoxide and significant DNA damage. Apoptosis followed these events. Taken together, the data suggest that diazeniumdiolate-based NO-releasing prodrugs may have application as a personalized therapy for lung cancers characterized by high levels of ROS/RNS. PRX1 and OGG1 proteins, which can be easily measured, could function as biomarkers for identifying tumors sensitive to the therapy.

The evolution and expression of the snaR family of small non-coding RNAs

PubMed Central

Parrott, Andrew M.; Tsai, Michael; Batchu, Priyanka; Ryan, Karen; Ozer, Harvey L.; Tian, Bin; Mathews, Michael B.

2011-01-01

We recently identified the snaR family of small non-coding RNAs that associate in vivo with the nuclear factor 90 (NF90/ILF3) protein. The major human species, snaR-A, is an RNA polymerase III transcript with restricted tissue distribution and orthologs in chimpanzee but not rhesus macaque or mouse. We report their expression in human tissues and their evolution in primates. snaR genes are exclusively in African Great Apes and some are unique to humans. Two novel families of snaR-related genetic elements were found in primates: CAS (catarrhine ancestor of snaR), limited to Old World Monkeys and apes; and ASR (Alu/snaR-related), present in all monkeys and apes. ASR and CAS appear to have spread by retrotransposition, whereas most snaR genes have spread by segmental duplication. snaR-A and snaR-G2 are differentially expressed in discrete regions of the human brain and other tissues, notably including testis. snaR-A is up-regulated in transformed and immortalized human cells, and is stably bound to ribosomes in HeLa cells. We infer that snaR evolved from the left monomer of the primate-specific Alu SINE family via ASR and CAS in conjunction with major primate speciation events, and suggest that snaRs participate in tissue- and species-specific regulation of cell growth and translation. PMID:20935053

Preclinical Testing of Antihuman CD28 Fab' Antibody in a Novel Nonhuman Primate Small Animal Rodent Model of Xenogenic Graft-Versus-Host Disease.

PubMed

Hippen, Keli L; Watkins, Benjamin; Tkachev, Victor; Lemire, Amanda M; Lehnen, Charles; Riddle, Megan J; Singh, Karnail; Panoskaltsis-Mortari, Angela; Vanhove, Bernard; Tolar, Jakub; Kean, Leslie S; Blazar, Bruce R

2016-12-01

Graft-versus-host disease (GVHD) is a severe complication of hematopoietic stem cell transplantation. Current therapies to prevent alloreactive T cell activation largely cause generalized immunosuppression and may result in adverse drug, antileukemia and antipathogen responses. Recently, several immunomodulatory therapeutics have been developed that show efficacy in maintaining antileukemia responses while inhibiting GVHD in murine models. To analyze efficacy and better understand immunological tolerance, escape mechanisms, and side effects of clinical reagents, testing of species cross-reactive human agents in large animal GVHD models is critical. We have previously developed and refined a nonhuman primate (NHP) large animal GVHD model. However, this model is not readily amenable to semi-high throughput screening of candidate clinical reagents. Here, we report a novel, optimized NHP xenogeneic GVHD (xeno-GVHD) small animal model that recapitulates many aspects of NHP and human GVHD. This model was validated using a clinically available blocking, monovalent anti-CD28 antibody (FR104) whose effects in a human xeno-GVHD rodent model are known. Because human-reactive reagents may not be fully cross-reactive or effective in vivo on NHP immune cells, this NHP xeno-GVHD model provides immunological insights and direct testing on NHP-induced GVHD before committing to the intensive NHP studies that are being increasingly used for detailed evaluation of new immune therapeutic strategies before human trials.

Developing Novel Therapeutic Approaches in Small Cell Lung Carcinoma Using Genetically Engineered Mouse Models and Human Circulating Tumor Cells

DTIC Science & Technology

2016-12-01

developed expertise in live animal imaging to enable monitoring to tumors over time in these models. We have initiated treatment studies with chemotherapy...requested on 9/22/14 and reported in our first annual report. Significant changes in use or care of human subjects, vertebrate animals ...biohazards and/or select agents We have no additional changes to make in use of vertebrate animals , biohazards and/or select reagents beyond what was

Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis

PubMed Central

1992-01-01

During human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4+ population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4+ cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4+ T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activation-induced cell death, suggesting a mechanism for CD4+ T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity. PMID:1402655

Antiviral CD8+ T Cells Restricted by Human Leukocyte Antigen Class II Exist during Natural HIV Infection and Exhibit Clonal Expansion.

PubMed

Ranasinghe, Srinika; Lamothe, Pedro A; Soghoian, Damien Z; Kazer, Samuel W; Cole, Michael B; Shalek, Alex K; Yosef, Nir; Jones, R Brad; Donaghey, Faith; Nwonu, Chioma; Jani, Priya; Clayton, Gina M; Crawford, Frances; White, Janice; Montoya, Alana; Power, Karen; Allen, Todd M; Streeck, Hendrik; Kaufmann, Daniel E; Picker, Louis J; Kappler, John W; Walker, Bruce D

2016-10-18

CD8 + T cell recognition of virus-infected cells is characteristically restricted by major histocompatibility complex (MHC) class I, although rare examples of MHC class II restriction have been reported in Cd4-deficient mice and a macaque SIV vaccine trial using a recombinant cytomegalovirus vector. Here, we demonstrate the presence of human leukocyte antigen (HLA) class II-restricted CD8 + T cell responses with antiviral properties in a small subset of HIV-infected individuals. In these individuals, T cell receptor β (TCRβ) analysis revealed that class II-restricted CD8 + T cells underwent clonal expansion and mediated killing of HIV-infected cells. In one case, these cells comprised 12% of circulating CD8 + T cells, and TCRα analysis revealed two distinct co-expressed TCRα chains, with only one contributing to binding of the class II HLA-peptide complex. These data indicate that class II-restricted CD8 + T cell responses can exist in a chronic human viral infection, and may contribute to immune control. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

MicroRNA-1285-5p influences the proliferation and metastasis of non-small-cell lung carcinoma cells via downregulating CDH1 and Smad4.

PubMed

Zhou, Shixia; Zhang, Zhongmian; Zheng, Pengyuan; Zhao, Wenchao; Han, Na

2017-06-01

Abnormal expression of microRNAs has been reported to regulate gene expression and cancer cell growth, invasion, and migration. Recently, upregulation of hsa-miR-1285 was demonstrated in bronchoalveolar lavage fluid samples from patients with lung cancer and downregulation in plasma level of stage-I lung cancer patients. However, the function and the underlying mechanism of miR-1285 in non-small-cell lung carcinoma have not been elucidated. In this study, we found that miR-1285-5p, the mature form of miR-1285, was significantly upregulated in human non-small-cell lung carcinoma cell lines A549 and SK-MES-1. Additionally, cells transfected with the miR-1285-5p inhibitor LV-anti-miR-1285-5p demonstrated significantly inhibited proliferation and invasion and depressed migration. Further analysis demonstrated that the miR-1285-5p precursor LV-miR-1285-5p attenuated the expression of Smad4 and cadherin-1 (CDH1) but that LV-anti-miR-1285-5p showed opposite results. A luciferase reporter assay confirmed that miR-1285-5p targeted Smad4 and CDH1. Mechanism analyses revealed that silence of Smad4 and CDH1 significantly attenuated the inhibitory effects of LV-anti-miR-1285-5p on non-small-cell lung carcinoma growth and invasion. Taken together, our data suggest that miR-1285-5p functions as a tumor promoter in the development of non-small-cell lung carcinoma by targeting Smad4 and CDH1, indicating a novel therapeutic strategy for non-small-cell lung carcinoma patients.

Generation of human secondary cardiospheres as a potent cell processing strategy for cell-based cardiac repair.

PubMed

Cho, Hyun-Jai; Lee, Ho-Jae; Chung, Yeon-Ju; Kim, Ju-Young; Cho, Hyun-Ju; Yang, Han-Mo; Kwon, Yoo-Wook; Lee, Hae-Young; Oh, Byung-Hee; Park, Young-Bae; Kim, Hyo-Soo

2013-01-01

Cell therapy is a promising approach for repairing damaged heart. However, there are large rooms to be improved in therapeutic efficacy. We cultured a small quantity (5-10 mg) of heart biopsy tissues from 16 patients who received heart transplantation. We produced primary and secondary cardiospheres (CSs) using repeated three-dimensional culture strategy and characterized the cells. Approximately 5000 secondary CSs were acquired after 45 days. Genetic analysis confirmed that the progenitor cells in the secondary CSs originated from the innate heart, but not from extra-cardiac organs. The expressions of Oct4 and Nanog were significantly induced in secondary CSs compared with adherent cells derived from primary CSs. Those expressions in secondary CSs were higher in a cytokine-deprived medium than in a cytokine-supplemented one, suggesting that formation of the three-dimensional structure was important to enhance stemness whereas supplementation with various cytokines was not essential. Signal blocking experiments showed that the ERK and VEGF pathways are indispensable for sphere formation. To optimize cell processing, we compared four different methods of generating spheres. Method based on the hanging-drop or AggreWell™ was superior to that based on the poly-d-lysine-coated dish or Petri dish with respect to homogeneity of the product, cellular potency and overall simplicity of the process. When transplanted into the ischemic myocardium of immunocompromised mice, human secondary CSs differentiated into cardiomyocytes and endothelial cells. These results demonstrate that generation of secondary CSs from a small quantity of adult human cardiac tissue is a feasible and effective cell processing strategy to improve the therapeutic efficacy of cell therapy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model.

PubMed

Drabsch, Yvette; He, Shuning; Zhang, Long; Snaar-Jagalska, B Ewa; ten Dijke, Peter

2013-11-07

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

The mechanisms for lung cancer risk of PM2.5 : Induction of epithelial-mesenchymal transition and cancer stem cell properties in human non-small cell lung cancer cells.

PubMed

Wei, Hongying; Liang, Fan; Cheng, Wei; Zhou, Ren; Wu, Xiaomeng; Feng, Yan; Wang, Yan

2017-11-01

Fine particulate matter (PM 2.5 ) is a major component of air pollutions that are closely associated with increased risk of lung cancer. However, the role of PM 2.5 in the etiology of lung cancer is largely unknown. In this study, we performed acute (24 hours) and chronic (five passages) exposure models to investigate the carcinogenetic mechanisms of PM 2.5 by targeting the induction of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) properties in human non-small cell lung cancer cell line A549. We found that both acute and chronic PM 2.5 exposure enhanced cell migration and invasion, decreased mRNA expression of epithelial markers and increased mRNA expression of mesenchymal markers. Chronic PM 2.5 exposure further induced notable EMT morphology and CSC properties, indicating the developing process of cell malignant behaviors from acute to chronic PM 2.5 exposure. CSC properties induced by chronic PM 2.5 exposure characterized with increased cell-surface markers (CD44, ABCG2), self-renewal genes (SOX2 and OCT4), side population cells and neoplastic capacity. Furthermore, the levels of three stemness-associated microRNAs, Let-7a, miR-16 and miR-34a, were found to be significantly downregulated by chronic PM 2.5 exposure, with microarray data analysis from TCGA database showing their lower expression in human lung adenocarcinoma tissues than that in the adjacent normal lung tissues. These data revealed that the induction of EMT and CSC properties were involved in the lung cancer risk of PM 2.5 , and implicated CSC properties and related microRNAs as possible biomarkers for carcinogenicity prediction of PM 2.5 . © 2017 Wiley Periodicals, Inc.

Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study.

PubMed

Bong, Ivyna Pau Ni; Ng, Ching Ching; Fakiruddin, Shaik Kamal; Lim, Moon Nian; Zakaria, Zubaidah

2016-11-10

Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the functions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM.

Small interfering RNA-mediated silencing of nicotinamide phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) induce growth inhibition and apoptosis in human multiple myeloma cells: A preliminary study

PubMed Central

Bong, Ivyna Pau Ni; Ng, Ching Ching; Fakiruddin, Shaik Kamal; Lim, Moon Nian; Zakaria, Zubaidah

2016-01-01

Multiple myeloma (MM) is a malignancy of B lymphocytes or plasma cells. Our array-based comparative genomic hybridization findings revealed chromosomal gains at 7q22.3 and 1q42.3, where nicotinamide (NAM) phosphoribosyltransferase (NAMPT) and lysosomal trafficking regulator (LYST) genes are localized, respectively. This led us to further study the fprotein expression in unctions of these genes in myeloma cells. NAMPT is a key enzyme involved in nicotinamide adenine dinucleotide salvage pathway, and it is frequently overexpressed in human cancers. In contrast, little is known about the function of LYST in cancer. The expression of LYST is shown to affect lysosomal size, granule size, and autophagy in human cells. In this study, the effects of small interfering RNA (siRNA)-mediated silencing of NAMPT and LYST on cell proliferation and apoptosis were evaluated in RPMI 8226 myeloma cells. Transfection efficiencies were determined by quantitative real time reverse transcriptase PCR. Cell proliferation was determined using MTT assay, while apoptosis was analyzed with flow cytometry using Annexin V-fluorescein isothiocyanate/propidium iodide assay. The NAMPT protein expression in siRNA-treated cells was estimated by enzyme-linked immunosorbent assay. Our results showed that NAMPT and LYST were successfully knockdown by siRNA transfection (p < 0.05). NAMPT or LYST gene silencing significantly inhibited cell proliferation and induced apoptosis in RPMI 8226 cells (p < 0.05). Silencing of NAMPT gene also decreased NAMPT protein levels (p < 0.01). Our study demonstrated that NAMPT and LYST play pivotal roles in the molecular pathogenesis of MM. This is the first report describing the possible functions of LYST in myelomagenesis and its potential role as a therapeutic target in MM. PMID:27754828

Efficacy of ONC201 in Desmoplastic Small Round Cell Tumor.

PubMed

Hayes-Jordan, Andrea A; Ma, Xiao; Menegaz, Brian A; Lamhamedi-Cherradi, Salah-Eddine; Kingsley, Charles V; Benson, Jalen A; Camacho, Pamela E; Ludwig, Joseph A; Lockworth, Cynthia R; Garcia, Gloria E; Craig, Suzanne L

2018-05-01

Desmoplastic Small Round Cell Tumor (DSRCT) is a rare sarcoma tumor of adolescence and young adulthood, which harbors a recurrent chromosomal translocation between the Ewing's sarcoma gene (EWSR1) and the Wilms' tumor suppressor gene (WT1). Patients usually develop multiple abdominal tumors with liver and lymph node metastasis developing later. Survival is poor using a multimodal therapy that includes chemotherapy, radiation and surgical resection, new therapies are needed for better management of DSRCT. Triggering cell apoptosis is the scientific rationale of many cancer therapies. Here, we characterized for the first time the expression of pro-apoptotic receptors, tumor necrosis-related apoptosis-inducing ligand receptors (TRAILR1-4) within an established human DSRCT cell line and clinical samples. The molecular induction of TRAIL-mediated apoptosis using agonistic small molecule, ONC201 in vitro cell-based proliferation assay and in vivo novel orthotopic xenograft animal models of DSRCT, was able to inhibit cell proliferation that was associated with caspase activation, and tumor growth, indicating that a cell-based delivery of an apoptosis-inducing factor could be relevant therapeutic agent to control DSRCT. Copyright © 2018. Published by Elsevier Inc.

Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis

PubMed Central

Yang, Yi; Zhao, Yi; Ai, Xinghao; Cheng, Baijun; Lu, Shun

2014-01-01

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells. PMID:25674209

A positive circuit of VEGF increases Glut-1 expression by increasing HIF-1α gene expression in human retinal endothelial cells.

PubMed

Choi, Yoon Kyung

2017-12-01

Treatment of human retinal microvascular endothelial cells (HRMECs) with vascular endothelial growth factor 165 (VEGF 165 ) increased hypoxia-inducible factor 1α (HIF-1α), VEGF, and glucose transporter 1 (Glut-1) mRNA expression and Glut-1 protein localization to the membrane. In contrast, treatment of human retinal pigment epithelium cells with VEGF 165 did not induce HIF-1α, VEGF, and Glut-1 gene expression. Microvascular endothelial cells are surrounded by astrocytic end feet in the retina. Astrocyte-derived A-kinase anchor protein 12 overexpression during hypoxia downregulated VEGF secretion, and this conditioned medium reduced VEGF and Glut-1 expression in HRMECs, suggesting that communications between astrocytes and endothelial cells may be the determinants of the blood vessel network. In HRMECs, HIF-1α small interfering RNA transfection blocked the VEGF 165 -mediated increase in VEGF and Glut-1 gene expression. Inhibition of protein kinase C (PKC) with inhibitor GF109203X or with a small interfering RNA targeting PKCζ attenuated the VEGF 165 -induced Glut-1 protein expression and VEGF and Glut-1 mRNA expression. In addition, results of an immunoprecipitation assay imply an interaction between VEGF receptor 2 (VEGFR2) and PKCζ in HRMECs. Therefore, VEGF secretion by hypoxic astrocytes may upregulate HIF-1α gene expression, inducing VEGF and Glut-1 expression via the VEGFR2-PKCζ axis in HRMECs.

Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

PubMed

Benoit, Yannick D; Mitchell, Ryan R; Risueño, Ruth M; Orlando, Luca; Tanasijevic, Borko; Boyd, Allison L; Aslostovar, Lili; Salci, Kyle R; Shapovalova, Zoya; Russell, Jennifer; Eguchi, Masakatsu; Golubeva, Diana; Graham, Monica; Xenocostas, Anargyros; Trus, Michael R; Foley, Ronan; Leber, Brian; Collins, Tony J; Bhatia, Mickie

2017-07-20

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/β-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/β-catenin signaling within CSCs. Disruption of CBP-β-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Isolation and gene expression analysis of single potential human spermatogonial stem cells.

PubMed

von Kopylow, K; Schulze, W; Salzbrunn, A; Spiess, A-N

2016-04-01

It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis. We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR. The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1). We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining. Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable. The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design. The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features. The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Viral Oncology: Molecular Biology and Pathogenesis

PubMed Central

Mui, Uyen Ngoc; Haley, Christopher T.; Tyring, Stephen K.

2017-01-01

Oncoviruses are implicated in approximately 12% of all human cancers. A large number of the world’s population harbors at least one of these oncoviruses, but only a small proportion of these individuals go on to develop cancer. The interplay between host and viral factors is a complex process that works together to create a microenvironment conducive to oncogenesis. In this review, the molecular biology and oncogenic pathways of established human oncoviruses will be discussed. Currently, there are seven recognized human oncoviruses, which include Epstein-Barr Virus (EBV), Human Papillomavirus (HPV), Hepatitis B and C viruses (HBV and HCV), Human T-cell lymphotropic virus-1 (HTLV-1), Human Herpesvirus-8 (HHV-8), and Merkel Cell Polyomavirus (MCPyV). Available and emerging therapies for these oncoviruses will be mentioned. PMID:29186062

Human immunophenotyping via low-variance, low-bias, interpretive regression modeling of small, wide data sets: Application to aging and immune response to influenza vaccination.

PubMed

Holmes, Tyson H; He, Xiao-Song

2016-10-01

Small, wide data sets are commonplace in human immunophenotyping research. As defined here, a small, wide data set is constructed by sampling a small to modest quantity n,1

Human Immunophenotyping via Low-Variance, Low-Bias, Interpretive Regression Modeling of Small, Wide Data Sets: Application to Aging and Immune Response to Influenza Vaccination

PubMed Central

Holmes, Tyson H.; He, Xiao-Song

2016-01-01

Small, wide data sets are commonplace in human immunophenotyping research. As defined here, a small, wide data set is constructed by sampling a small to modest quantity n, 1 < n < 50, of human participants for the purpose of estimating many parameters p, such that n < p < 1,000. We offer a set of prescriptions that are designed to facilitate low-variance (i.e. stable), low-bias, interpretive regression modeling of small, wide data sets. These prescriptions are distinctive in their especially heavy emphasis on minimizing use of out-of-sample information for conducting statistical inference. That allows the working immunologist to proceed without being encumbered by imposed and often untestable statistical assumptions. Problems of unmeasured confounders, confidence-interval coverage, feature selection, and shrinkage/denoising are defined clearly and treated in detail. We propose an extension of an existing nonparametric technique for improved small-sample confidence-interval tail coverage from the univariate case (single immune feature) to the multivariate (many, possibly correlated immune features). An important role for derived features in the immunological interpretation of regression analyses is stressed. Areas of further research are discussed. Presented principles and methods are illustrated through application to a small, wide data set of adults spanning a wide range in ages and multiple immunophenotypes that were assayed before and after immunization with inactivated influenza vaccine (IIV). Our regression modeling prescriptions identify some potentially important topics for future immunological research. 1) Immunologists may wish to distinguish age-related differences in immune features from changes in immune features caused by aging. 2) A form of the bootstrap that employs linear extrapolation may prove to be an invaluable analytic tool because it allows the working immunologist to obtain accurate estimates of the stability of immune parameter estimates with a bare minimum of imposed assumptions. 3) Liberal inclusion of immune features in phenotyping panels can facilitate accurate separation of biological signal of interest from noise. In addition, through a combination of denoising and potentially improved confidence interval coverage, we identify some candidate immune correlates (frequency of cell subset and concentration of cytokine) with B cell response as measured by quantity of IIV-specific IgA antibody-secreting cells and quantity of IIV-specific IgG antibody-secreting cells. PMID:27196789

Progressive changes in non-coding RNA profile in leucocytes with age

PubMed Central

Muñoz-Culla, Maider; Irizar, Haritz; Gorostidi, Ana; Alberro, Ainhoa; Osorio-Querejeta, Iñaki; Ruiz-Martínez, Javier; Olascoaga, Javier; de Munain, Adolfo López; Otaegui, David

2017-01-01

It has been observed that immune cell deterioration occurs in the elderly, as well as a chronic low-grade inflammation called inflammaging. These cellular changes must be driven by numerous changes in gene expression and in fact, both protein-coding and non-coding RNA expression alterations have been observed in peripheral blood mononuclear cells from elder people. In the present work we have studied the expression of small non-coding RNA (microRNA and small nucleolar RNA -snoRNA-) from healthy individuals from 24 to 79 years old. We have observed that the expression of 69 non-coding RNAs (56 microRNAs and 13 snoRNAs) changes progressively with chronological age. According to our results, the age range from 47 to 54 is critical given that it is the period when the expression trend (increasing or decreasing) of age-related small non-coding RNAs is more pronounced. Furthermore, age-related miRNAs regulate genes that are involved in immune, cell cycle and cancer-related processes, which had already been associated to human aging. Therefore, human aging could be studied as a result of progressive molecular changes, and different age ranges should be analysed to cover the whole aging process. PMID:28448962

The Immunomodulatory Small Molecule Imiquimod Induces Apoptosis in Devil Facial Tumour Cell Lines.

PubMed

Patchett, Amanda L; Darby, Jocelyn M; Tovar, Cesar; Lyons, A Bruce; Woods, Gregory M

2016-01-01

The survival of the Tasmanian devil (Sarcophilus harrisii) is threatened by devil facial tumour disease (DFTD). This transmissible cancer is usually fatal, and no successful treatments have been developed. In human studies, the small immunomodulatory molecule imiquimod is a successful immunotherapy, activating anti-tumour immunity via stimulation of toll-like receptor-7 (TLR7) signaling pathways. In addition, imiquimod is a potent inducer of apoptosis in human tumour cell lines via TLR7 independent mechanisms. Here we investigate the potential of imiquimod as a DFTD therapy through analysis of treated DFTD cell lines and Tasmanian devil fibroblasts. WST-8 proliferation assays and annexin V apoptosis assays were performed to monitor apoptosis, and changes to the expression of pro- and anti-apoptotic genes were analysed using qRT-PCR. Our results show that DFTD cell lines, but not Tasmanian devil fibroblasts, are sensitive to imiquimod-induced apoptosis in a time and concentration dependent manner. Induction of apoptosis was accompanied by down-regulation of the anti-apoptotic BCL2 and BCLXL genes, and up-regulation of the pro-apoptotic BIM gene. Continuous imiquimod treatment was required for these effects to occur. These results demonstrate that imiquimod can deregulate DFTD cell growth and survival in direct and targeted manner. In vivo, this may increase DFTD vulnerability to imiquimod-induced TLR7-mediated immune responses. Our findings have improved the current knowledge of imiquimod action in tumour cells for application to both DFTD and human cancer therapy.

Small glutamine-rich tetratricopeptide repeat-containing protein alpha is present in human ovaries but may not be differentially expressed in relation to polycystic ovary syndrome.

PubMed

Butler, Miriam S; Yang, Xing; Ricciardelli, Carmela; Liang, Xiaoyan; Norman, Robert J; Tilley, Wayne D; Hickey, Theresa E

2013-06-01

To evaluate the expression and function of small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), an androgen receptor (AR) molecular chaperone, in human ovarian tissues. Examine the effect of SGTA on AR subcellular localization in granulosa tumor cells (KGN) and SGTA expression in ovarian tissues. University-based research laboratory. Archived tissues from premenopausal women and granulosa cells from infertile women receiving assisted reproduction. None. AR subcellular localization and SGTA protein or mRNA levels. SGTA and AR proteins were expressed in the cytoplasm of KGN cells and exposure to androgen stimulated AR nuclear localization. SGTA protein knockdown increased AR nuclear localization at low (0-0.1 nmol/L) but not high (1-10 nmol/L) concentrations of androgen hormone. In ovarian tissues, SGTA was localized to the cytoplasm of granulosa cells at all stages of folliculogenesis and in thecal cells of antral follicles. SGTA protein levels were similar when comparing primordial and primary follicles within core biopsies (n = 40) from women with and without polycystic ovary syndrome (PCOS). Likewise, SGTA mRNA levels were not significantly different in granulosa cells from preovulatory follicles after hyperstimulation of women with and without PCOS. SGTA is present in human ovaries and has the potential to modulate AR signalling, but it may not be differentially expressed in PCOS. Copyright © 2013 American Society for Reproductive Medicine. All rights reserved.

MicroRNA-300 targets hypoxia inducible factor-3 alpha to inhibit tumorigenesis of human non-small cell lung cancer.

PubMed

Zhang, Y; Guo, Y; Yang, C; Zhang, S; Zhu, X; Cao, L; Nie, W; Yu, H

2017-01-01

Non-small cell lung cancer (NSCLC) is one of the most deadly human cancers. MicroRNA-300 acts as both tumor promoter and suppressor in different types of cancer. Here, we try to identify the function of microRNA-300 in human NSCLC. We compared MicroRNA-300 levels between tumor tissues versus paired adjacent non-tumor lung tissues from NSCLC patients, and in NSCLC versus normal lung cell lines. Effects of microRNA-300 on cell proliferation, invasion and migration were examined in vitro, and on tumor growth in vivo using a xenograft mouse model. Potential mRNA targets of microRNA-300 were predicted and underlying mechanism was explored. MicroRNA-300 expression was lower in both NSCLC tissues and cell lines. Overexpression of microRNA-300 inhibited proliferation, invasion and migration of NSCLC cells in vitro, and tumor growth in vivo. MicroRNA-300 could directly bind to the 3'-UTR of hypoxia inducible factor-3 alpha (HIF3α) mRNA, and inhibit both its mRNA and protein expressions. Restoring HIF3α expression could rescue the inhibitory effects of microRNA-300 on tumorigenesis of NSCLC both in vitro and in vivo. MicroRNA-300 is a tumor suppressor microRNA in NSCLC by downregulating HIF3α expression. Both microRNA-300 and HIF3α may serve as potential therapeutic targets in NSCLC treatment.

TNF-α enhancement of CD62E mediates adhesion of non-small cell lung cancer cells to brain endothelium via CD15 in lung-brain metastasis.

PubMed

Jassam, Samah A; Maherally, Zaynah; Smith, James R; Ashkan, Keyoumars; Roncaroli, Federico; Fillmore, Helen L; Pilkington, Geoffrey J

2016-05-01

CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non-small cell lung cancer (NSCLC). CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry. CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies. This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.

TNF-α enhancement of CD62E mediates adhesion of non–small cell lung cancer cells to brain endothelium via CD15 in lung-brain metastasis

PubMed Central

Jassam, Samah A.; Maherally, Zaynah; Smith, James R.; Ashkan, Keyoumars; Roncaroli, Federico; Fillmore, Helen L.; Pilkington, Geoffrey J.

2016-01-01

Background CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non–small cell lung cancer (NSCLC). Methods CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry. Results CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies. Conclusion This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E. PMID:26472821

Merkel Cell Polyomavirus: A Newly Discovered Human Virus with Oncogenic Potential

PubMed Central

Spurgeon, Megan E.; Lambert, Paul F.

2012-01-01

A marked escalation in the rate of discovery of new types of human polyomavirus has occurred over the last five years largely owing to recent technological advances in their detection. Among the newly discovered viruses, Merkel Cell Polyomavirus (MCPyV or MCV) has gained the most attention due to its link with a rare human cancer. Infection with MCPyV is common in the human population, and the virus is detected in several anatomical locations, but most frequently in skin. Study of MCPyV molecular virology has been complicated by the lack of straightforward cell culture models, but recent in vitro studies are making strides towards understanding the virus life cycle, its cellular tropism, and mode of transmission. While MCPyV shares several traditional traits with other human polyomaviruses, the burst of research since its discovery reveals insight into a virus with many unique genetic and mechanistic features. The evidence for a causal link between MCPyV and the rare neuroendocrine cancer, Merkel Cell Carcinoma (MCC), is compelling. A majority of MCCs contain clonally integrated viral DNA, express viral T antigen transcripts and protein, and exhibit an addiction to the viral large T and small t antigen oncoproteins. The MCPyV large T antigen contains MCC tumor-specific mutations that ablate its replication capacity but preserve its oncogenic functions, and the small t antigen promotes an environment favorable for cap-dependent translation. The mechanisms of MCPyV-induced transformation have not been fully elucidated, but the likely etiological role of this new polyomavirus in human cancer provides a strong opportunity to expand knowledge of virus-host interactions and viral oncology. PMID:23217622

CD36- and GPR120-mediated Ca²⁺ signaling in human taste bud cells mediates differential responses to fatty acids and is altered in obese mice.

PubMed

Ozdener, Mehmet Hakan; Subramaniam, Selvakumar; Sundaresan, Sinju; Sery, Omar; Hashimoto, Toshihiro; Asakawa, Yoshinori; Besnard, Philippe; Abumrad, Nada A; Khan, Naim Akhtar

2014-04-01

It is important to increase our understanding of gustatory detection of dietary fat and its contribution to fat preference. We studied the roles of the fat taste receptors CD36 and GPR120 and their interactions via Ca(2+) signaling in fungiform taste bud cells (TBC). We measured Ca(2+) signaling in human TBC, transfected with small interfering RNAs against messenger RNAs encoding CD36 and GPR120 (or control small interfering RNAs). We also studied Ca(2+) signaling in TBC from CD36(-/-) mice and from wild-type lean and obese mice. Additional studies were conducted with mouse enteroendocrine cell line STC-1 that express GPR120 and stably transfected with human CD36. We measured release of serotonin and glucagon-like peptide-1 from human and mice TBC in response to CD36 and GPR120 activation. High concentrations of linoleic acid induced Ca(2+) signaling via CD36 and GPR120 in human and mice TBC, as well as in STC-1 cells, and low concentrations induced Ca(2+) signaling via only CD36. Incubation of human and mice fungiform TBC with lineoleic acid down-regulated CD36 and up-regulated GPR120 in membrane lipid rafts. Obese mice had decreased spontaneous preference for fat. Fungiform TBC from obese mice had reduced Ca(2+) and serotonin responses, but increased release of glucagon-like peptide-1, along with reduced levels of CD36 and increased levels of GPR120 in lipid rafts. CD36 and GPR120 have nonoverlapping roles in TBC signaling during orogustatory perception of dietary lipids; these are differentially regulated by obesity. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Estimates of cellular mutagenesis from cosmic rays

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Wilson, John W.

1994-01-01

A parametric track structure model is used to estimate the cross section as a function of particle velocity and charge for mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in human fibroblast cell cultures. Experiments that report the fraction of mutations per surviving cell for human lung and skin fibroblast cells indicate small differences in the mutation cross section for these two cell lines when differences in inactivation rates between these cell lines are considered. Using models of cosmic ray transport, the mutation rate at the HGPRT locus is estimated for cell cultures in space flight and rates of about 2 to 10 x 10(exp -6) per year are found for typical spacecraft shielding. A discussion of how model assumptions may alter the predictions is also presented.

Invariant natural killer T cells trigger adaptive lymphocytes to churn up bile.

PubMed

Joyce, Sebastian; Van Kaer, Luc

2008-05-15

How innate immune response causes autoimmunity has remained an enigma. In this issue of Cell Host & Microbe, Mattner et al. demonstrate that invariant natural killer T cells activated by the mucosal commensal Novosphingobium aromaticivorans precipitate chronic T cell-mediated autoimmunity against small bile ducts that mirrors human primary biliary cirrhosis. These findings provide a mechanistic understanding of the role of innate immunity toward a microbe in the development of autoimmunity.

Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals.

PubMed

Qiu, Yang; Xu, Yanpeng; Zhang, Yao; Zhou, Hui; Deng, Yong-Qiang; Li, Xiao-Feng; Miao, Meng; Zhang, Qiang; Zhong, Bo; Hu, Yuanyang; Zhang, Fu-Chun; Wu, Ligang; Qin, Cheng-Feng; Zhou, Xi

2017-06-20

RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals. Copyright © 2017 Elsevier Inc. All rights reserved.

Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle.

PubMed

Yim, Peter D; Gallos, George; Perez-Zoghbi, Jose F; Trice, Jacquelyn; Zhang, Yi; Siviski, Matthew; Sonett, Joshua; Emala, Charles W

2013-01-01

Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K(+) channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders.

Pathobiologic Roles of Epstein–Barr Virus-Encoded MicroRNAs in Human Lymphomas

PubMed Central

Navari, Mohsen; Etebari, Maryam; Ibrahimi, Mostafa; Leoncini, Lorenzo

2018-01-01

Epstein–Barr virus (EBV) is a human γ-herpesvirus implicated in several human malignancies, including a wide range of lymphomas. Several molecules encoded by EBV in its latent state are believed to be related to EBV-induced lymphomagenesis, among which microRNAs—small RNAs with a posttranscriptional regulating role—are of great importance. The genome of EBV encodes 44 mature microRNAs belonging to two different classes, including BamHI-A rightward transcript (BART) and Bam HI fragment H rightward open reading frame 1 (BHRF1), with different expression levels in different EBV latency types. These microRNAs might contribute to the pathogenetic effects exerted by EBV through targeting self mRNAs and host mRNAs and interfering with several important cellular mechanisms such as immunosurveillance, cell proliferation, and apoptosis. In addition, EBV microRNAs can regulate the surrounding microenvironment of the infected cells through exosomal transportation. Moreover, these small molecules could be potentially used as molecular markers. In this review, we try to present an updated and extensive view of the role of EBV-encoded miRNAs in human lymphomas. PMID:29649101

The cell size and distribution of adipocytes from subcutaneous and visceral fat is associated with type 2 diabetes mellitus in humans.

PubMed

Fang, Lingling; Guo, Fangjian; Zhou, Lihua; Stahl, Richard; Grams, Jayleen

2015-01-01

Regional deposition of adipose tissue and adipocyte morphology may contribute to increased risk for insulin resistance. The aim of this study was to compare adipocyte cell size and size distribution from multiple fat depots and to determine the association with type 2 diabetes mellitus, anthropomorphic data, and subjects' metabolic profile. Clinical data and adipose tissue from subcutaneous fat, omentum, and mesentery were collected from 30 subjects with morbid obesity. Adipocytes were isolated by collagenase digestion and sized by microscopic measurement of cell diameter. Overall, adipocytes from subcutaneous fat were larger than those from omentum or mesentery. For the subcutaneous and omental fat depots, there was a significant increase in % small cells (14.9% vs 31.4%, p = 0 .006 and 14.0% vs 30.5%, p = 0 .015, respectively) and corresponding decrease in % large cells for nondiabetic vs diabetic patients. There was a similar trend for mesentery but it did not reach statistical significance (p = 0 .090). For omentum and mesentery, there was also a significant decrease in the diameter of the small cells. Fasting glucose was positively correlated with fraction of small cells in omentum and mesentery, and HbA1C was positively correlated with fraction of small cells in the omental fat depot. There was no correlation between large cell diameter with clinical parameters in any of the fat depots. These results indicate size distribution of adipocytes, specifically an increase in the fraction of small cells, is associated with the presence of type 2 diabetes mellitus.

The cell size and distribution of adipocytes from subcutaneous and visceral fat is associated with type 2 diabetes mellitus in humans

PubMed Central

Fang, Lingling; Guo, Fangjian; Zhou, Lihua; Stahl, Richard; Grams, Jayleen

2015-01-01

Aims/hypothesis: Regional deposition of adipose tissue and adipocyte morphology may contribute to increased risk for insulin resistance. The aim of this study was to compare adipocyte cell size and size distribution from multiple fat depots and to determine the association with type 2 diabetes mellitus, anthropomorphic data, and subjects' metabolic profile. Methods: Clinical data and adipose tissue from subcutaneous fat, omentum, and mesentery were collected from 30 subjects with morbid obesity. Adipocytes were isolated by collagenase digestion and sized by microscopic measurement of cell diameter. Results: Overall, adipocytes from subcutaneous fat were larger than those from omentum or mesentery. For the subcutaneous and omental fat depots, there was a significant increase in % small cells (14.9% vs 31.4%, p = 0 .006 and 14.0% vs 30.5%, p = 0 .015, respectively) and corresponding decrease in % large cells for nondiabetic vs diabetic patients. There was a similar trend for mesentery but it did not reach statistical significance (p = 0 .090). For omentum and mesentery, there was also a significant decrease in the diameter of the small cells. Fasting glucose was positively correlated with fraction of small cells in omentum and mesentery, and HbA1C was positively correlated with fraction of small cells in the omental fat depot. There was no correlation between large cell diameter with clinical parameters in any of the fat depots. Conclusions/interpretation: These results indicate size distribution of adipocytes, specifically an increase in the fraction of small cells, is associated with the presence of type 2 diabetes mellitus. PMID:26451283

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells

PubMed Central

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A.; de Boer, Jan; Watt, Fiona M.

2016-01-01

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation. PMID:26757610

Scalable topographies to support proliferation and Oct4 expression by human induced pluripotent stem cells.

PubMed

Reimer, Andreas; Vasilevich, Aliaksei; Hulshof, Frits; Viswanathan, Priyalakshmi; van Blitterswijk, Clemens A; de Boer, Jan; Watt, Fiona M

2016-01-13

It is well established that topographical features modulate cell behaviour, including cell morphology, proliferation and differentiation. To define the effects of topography on human induced pluripotent stem cells (iPSC), we plated cells on a topographical library containing over 1000 different features in medium lacking animal products (xeno-free). Using high content imaging, we determined the effect of each topography on cell proliferation and expression of the pluripotency marker Oct4 24 h after seeding. Features that maintained Oct4 expression also supported proliferation and cell-cell adhesion at 24 h, and by 4 days colonies of Oct4-positive, Sox2-positive cells had formed. Computational analysis revealed that small feature size was the most important determinant of pluripotency, followed by high wave number and high feature density. Using this information we correctly predicted whether any given topography within our library would support the pluripotent state at 24 h. This approach not only facilitates the design of substrates for optimal human iPSC expansion, but also, potentially, identification of topographies with other desirable characteristics, such as promoting differentiation.

QUANTITATION OF ABERRANT INTERLOCUS T-CELL RECEPTOR REARRANGEMENTS IN MOUSE THYMOCYTES AND THE EFFECT OF THE HERBICIDE 2,4- DICHLOROPHENOXYACETIC ACID

EPA Science Inventory

Quantitation of aberrant interlocus T-cell receptor rearrangements in mouse thymocytes and the effect of the herbicide 2,4- Dichlorophenoxyacetic acid

Small studies in human populations have suggested a correlation between the frequency of errors in antigen receptor gene a...

Small GTPases are involved in sprout formation in human granulosa lutein cells.

PubMed

Franz, Maximilian B; Daube, Stefanie; Keck, Christoph; Sator, Michael; Pietrowski, Detlef

2013-04-01

The corpus luteum (CL), develops from the ruptured follicle after gonadotropin stimulation. Based on intracellular reorganization of the cytoskeleton an human chorionic gonadotropin (hCG) dependent sprouting and migration of luteinizing granulosa cells (LGCs) and endothelial cells is observed. Rho-GTPases are shown to be key regulators of cytoskeletal restructuring. In the present study we analyzed the role of Rho-GTPases in the sprouting activity of LGCs. We used the Rho-GTPase-inhibitors Toxin A and -B and the Cdc42-activator Bradykinin in a LGC-spheroid sprouting assay to determine the effect of these modulators in LGCs. Toxin A and Toxin B reduces sprout formation in LGC spheroids. However, the reduction is less than in hCG treated cells. The usage of Bradykinin demonstrates both, a reduction of sprouts in untreated spheroids and an increase of sprouting in previous hCG treated spheroids. The presented results let us suggest that small Rho-GTPases may regulate the sprouting activity of LGCs after stimulation by hCG and that this mechanism may play a role in CL formation.

The role of CD133 in normal human prostate stem cells and malignant cancer-initiating cells.

PubMed

Vander Griend, Donald J; Karthaus, Wouter L; Dalrymple, Susan; Meeker, Alan; DeMarzo, Angelo M; Isaacs, John T

2008-12-01

Resolving the specific cell of origin for prostate cancer is critical to define rational targets for therapeutic intervention and requires the isolation and characterization of both normal human prostate stem cells and prostate cancer-initiating cells (CIC). Single epithelial cells from fresh normal human prostate tissue and prostate epithelial cell (PrEC) cultures derived from them were evaluated for the presence of subpopulations expressing stem cell markers and exhibiting stem-like growth characteristics. When epithelial cell suspensions containing cells expressing the stem cell marker CD133+ are inoculated in vivo, regeneration of stratified human prostate glands requires inductive prostate stromal cells. PrEC cultures contain a small subpopulation of CD133+ cells, and fluorescence-activated cell sorting-purified CD133+ PrECs self-renew and regenerate cell populations expressing markers of transit-amplifying cells (DeltaNp63), intermediate cells (prostate stem cell antigen), and neuroendocrine cells (CD56). Using a series of CD133 monoclonal antibodies, attachment and growth of CD133+ PrECs requires surface expression of full-length glycosylated CD133 protein. Within a series of androgen receptor-positive (AR+) human prostate cancer cell lines, CD133+ cells are present at a low frequency, self-renew, express AR, generate phenotypically heterogeneous progeny negative for CD133, and possess an unlimited proliferative capacity, consistent with CD133+ cells being CICs. Unlike normal adult prostate stem cells, prostate CICs are AR+ and do not require functional CD133. This suggests that (a) AR-expressing prostate CICs are derived from a malignantly transformed intermediate cell that acquires "stem-like activity" and not from a malignantly transformed normal stem cell and (b) AR signaling pathways are a therapeutic target for prostate CICs.

Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells.

PubMed

Mello, Paola de Andrade; Filippi-Chiela, Eduardo Cremonese; Nascimento, Jéssica; Beckenkamp, Aline; Santana, Danielle Bertodo; Kipper, Franciele; Casali, Emerson André; Nejar Bruno, Alessandra; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Wink, Marcia Rosângela; Lenz, Guido; Buffon, Andréia

2014-10-01

In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2 × 7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2 × 7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2 × 7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling-p53 increase, AMPK activation, and PARP cleavage-as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. © 2014 Mello et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Dimethyl sulfoxide (DMSO) exacerbates cisplatin-induced sensory hair cell death in zebrafish (Danio rerio).

PubMed

Uribe, Phillip M; Mueller, Melissa A; Gleichman, Julia S; Kramer, Matthew D; Wang, Qi; Sibrian-Vazquez, Martha; Strongin, Robert M; Steyger, Peter S; Cotanche, Douglas A; Matsui, Jonathan I

2013-01-01

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.

Dimethyl Sulfoxide (DMSO) Exacerbates Cisplatin-induced Sensory Hair Cell Death in Zebrafish (Danio rerio)

PubMed Central

Gleichman, Julia S.; Kramer, Matthew D.; Wang, Qi; Sibrian-Vazquez, Martha; Strongin, Robert M.; Steyger, Peter S.; Cotanche, Douglas A.; Matsui, Jonathan I.

2013-01-01

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO. PMID:23383324

Capsule-Like Safe Genetic Vectors - Cell-Penetrating Core-Shell Particles Selectively Release Functional Small RNA and Entrap its Encoding DNA.

PubMed

Yu, Han; Pan, Houwen Matthew; Evalin, Fnu; Trau, Dieter Wilhelm; Patzel, Volker

2018-06-05

The breakthrough of genetic therapy is set back by the lack of suitable genetic vector systems. We present the development of permeability-tunable, capsule-like, polymeric, micron-sized, core-shell particles for delivery of recombinant nucleic acids into target cells. These particles were demonstrated to effectively release rod-shaped small hairpin RNA and to selectively retain the RNA-encoding DNA template which was designed to form a bulky tripartite structure. Thus, they can serve as delivery vectors preloaded with cargo RNA or alternatively as RNA producing micro-bioreactors. The internalization of particles by human tissue culture cells inversely correlated with particle size and with the cell to particle ratio, though at a higher than stoichiometric excess of particles over cells, cell viability was impaired. Among primary human peripheral blood mononuclear cells, up to 50% of the monocytes displayed positive uptake of particles. Finally, these particles efficiently delivered siRNA into HEK293T cells triggering functional knockdown of the target gene lamin A/C. Particle-mediated knockdown was superior to that observed after conventional siRNA delivery via lipofection. Core-shell particles protect encapsulated nucleic acids from degradation and target cell genomes from direct contact with recombinant DNA, thus representing a promising delivery vector system that can be explored for genetic therapy and vaccination.

MiR-153 inhibits migration and invasion of human non-small-cell lung cancer by targeting ADAM19

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

Shan, Nianxi; Institute of Medical Sciences, Xiangya Hospital, Central South University, Changsha, Hunan 410008; Shen, Liangfang

Highlights: • Decreased miR-153 and up-regulated ADAM19 are correlated with NSCLC pathology. • MiR-153 inhibits the proliferation and migration and invasion of NSCLC cells in vitro. • ADAM19 is a direct target of miR-153. • ADAM19 is involved in miR-153-suppressed migration and invasion of NSCLC cells. - Abstract: MiR-153 was reported to be dysregulated in some human cancers. However, the function and mechanism of miR-153 in lung cancer cells remains unknown. In this study, we investigated the role of miR-153 in human non-small-cell lung cancer (NSCLC). Using qRT-PCR, we demonstrated that miR-153 was significantly decreased in clinical NSCLC tissues andmore » cell lines, and downregulation of miR-153 was significantly correlated with lymph node status. We further found that ectopic expression of miR-153 significantly inhibited the proliferation and migration and invasion of NSCLC cells in vitro, suggesting that miR-153 may be a novel tumor suppressor in NSCLC. Further integrated analysis revealed that ADAM19 is as a direct and functional target of miR-153. Luciferase reporter assay demonstrated that miR-153 directly targeted 3′UTR of ADAM19, and correlation analysis revealed an inverse correlation between miR-153 and ADAM19 mRNA levels in clinical NSCLC tissues. Knockdown of ADAM19 inhibited migration and invasion of NSCLC cells which was similar with effects of overexpression of miR-153, while overexpression of ADAM19 attenuated the function of miR-153 in NSCLC cells. Taken together, our results highlight the significance of miR-153 and ADAM19 in the development and progression of NSCLC.« less

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein

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

Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNAmore » expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.« less

Inhibition of connective tissue growth factor (CTGF/CCN2) expression decreases the survival and myogenic differentiation of human rhabdomyosarcoma cells.

PubMed

Croci, Stefania; Landuzzi, Lorena; Astolfi, Annalisa; Nicoletti, Giordano; Rosolen, Angelo; Sartori, Francesca; Follo, Matilde Y; Oliver, Noelynn; De Giovanni, Carla; Nanni, Patrizia; Lollini, Pier-Luigi

2004-03-01

Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

Carcinocythaemia (carcinoma cell leukaemia) in a dog: an acute leukaemia-like picture due to metastatic carcinoma.

PubMed

Amati, M; Miele, F; Avallone, G; Banco, B; Bertazzolo, W

2012-08-01

An eight-year-old entire female boxer was presented with a two-week history of anorexia and lethargy and two-day history of unilateral left epistaxis. Clinical findings and laboratory test results suggested disseminated intravascular coagulation. On blood smear evaluation, occasional large epithelioid-like unclassified cells were detected. Occasionally these cells were organised in small clusters. Bone marrow examination revealed a marked infiltration by a malignant population of the same epithelioid-like cells. The dog was euthanased because of the guarded prognosis. Following histology and immunohistochemistry, a widespread undifferentiated carcinoma of unknown primary origin was diagnosed. To the authors' knowledge, this is the first case of carcinoma cell leukaemia reported in a dog. Carcinoma cell leukaemia is a rare oncological condition previously described in humans, characterised by non-haematopoietic neoplastic cells in peripheral blood. © 2012 British Small Animal Veterinary Association.

Airway epithelial cell response to human metapneumovirus infection

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

Bao, X.; Liu, T.; Spetch, L.

2007-11-10

Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and typemore » I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.« less

Small organelle, big responsibility: the role of centrosomes in development and disease

PubMed Central

Chavali, Pavithra L.; Pütz, Monika; Gergely, Fanni

2014-01-01

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development. PMID:25047622

Improving the Safety of T Cell Therapies using an Inducible Caspase-9 Gene

PubMed Central

Zhou, Xiaoou; Brenner, Malcolm K.

2016-01-01

Adoptive transfer of T cells can be an effective anti-cancer treatment. However, uncontrolled or unpredictable immediate or persistent toxicities are a source of concern. The ability to conditionally eliminate aberrant cells in vivo therefore is becoming a critical step for the successful translation of this approach to the clinic. We review the evolution of safety systems, focusing on a suicide switch that can be expressed stably and efficiently in human T cells without impairing phenotype, function or antigen specificity. This system is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization in the presence of an otherwise bioinert small molecule drug. When exposed to the synthetic dimerizing drug, the inducible caspase 9 (iC9) becomes activated and leads to the rapid apoptosis of cells expressing this construct. We have demonstrated the clinical feasibility and efficacy of this approach after haploidentical hematopoietic stem cell transplant (haplo-HSCT). Here we review the benefits and limitations of the approach. PMID:27473568

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway.

PubMed

Zhao, Yana; Zhang, Shaojing; Wang, Pengfei; Fu, Shengnan; Wu, Di; Liu, Anjun

2017-12-01

Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.

Infrared Hollow Optical Fiber Probe for Localized Carbon Dioxide Measurement in Respiratory Tracts

PubMed Central

Katagiri, Takashi; Shibayama, Kyosuke; Iida, Takeru

2018-01-01

A real-time gas monitoring system based on optical absorption spectroscopy is proposed for localized carbon dioxide (CO2) measurement in respiratory tracts. In this system, a small gas cell is attached to the end of a hollow optical fiber that delivers mid-infrared light with small transmission loss. The diameters of the fiber and the gas cell are smaller than 1.2 mm so that the probe can be inserted into a working channel of common bronchoscopes. The dimensions of the gas cell are designed based on absorption spectra of CO2 standard gases in the 4.2 μm wavelength region, which are measured using a Fourier-transform infrared spectrometer. A miniature gas cell that is comprised of a stainless-steel tube with slots for gas inlet and a micro-mirror is fabricated. A compact probing system with a quantum cascade laser (QCL) light source is built using a gas cell with a hollow optical fiber for monitoring CO2 concentration. Experimental results using human breaths show the feasibility of the system for in-situ measurement of localized CO2 concentration in human airways. PMID:29584666

Screening Mammalian Cells on a Hydrogel: Functionalized Small Molecule Microarray.

PubMed

Zhu, Biwei; Jiang, Bo; Na, Zhenkun; Yao, Shao Q

2017-01-01

Mammalian cell-based microarray technology has gained wide attention, for its plethora of promising applications. The platform is able to provide simultaneous information on multiple parameters for a given target, or even multiple target proteins, in a complex biological system. Here we describe the preparation of mammalian cell-based microarrays using selectively captured of human prostate cancer cells (PC-3). This platform was then used in controlled drug release and measuring the associated drug effects on these cancer cells.

Minimum Transendothelial Electrical Resistance Thresholds for the Study of Small and Large Molecule Drug Transport in a Human in Vitro Blood-Brain Barrier Model.

PubMed

Mantle, Jennifer L; Min, Lie; Lee, Kelvin H

2016-12-05

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here, human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ω·cm 2 . By assessing the permeabilities of several known drugs, a benchmarking system to evaluate brain permeability of drugs was established. Furthermore, relationships between TEER and permeability to both small and large molecules were established, demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype, and the benchmarks established here are useful for assessing functionality of other in vitro BBB models.

Raman spectroscopy identifies radiation response in human non-small cell lung cancer xenografts

NASA Astrophysics Data System (ADS)

Harder, Samantha J.; Isabelle, Martin; Devorkin, Lindsay; Smazynski, Julian; Beckham, Wayne; Brolo, Alexandre G.; Lum, Julian J.; Jirasek, Andrew

2016-02-01

External beam radiation therapy is a standard form of treatment for numerous cancers. Despite this, there are no approved methods to account for patient specific radiation sensitivity. In this report, Raman spectroscopy (RS) was used to identify radiation-induced biochemical changes in human non-small cell lung cancer xenografts. Chemometric analysis revealed unique radiation-related Raman signatures that were specific to nucleic acid, lipid, protein and carbohydrate spectral features. Among these changes was a dramatic shift in the accumulation of glycogen spectral bands for doses of 5 or 15 Gy when compared to unirradiated tumours. When spatial mapping was applied in this analysis there was considerable variability as we found substantial intra- and inter-tumour heterogeneity in the distribution of glycogen and other RS spectral features. Collectively, these data provide unique insight into the biochemical response of tumours, irradiated in vivo, and demonstrate the utility of RS for detecting distinct radiobiological responses in human tumour xenografts.

B Lymphocyte intestinal homing in inflammatory bowel disease

PubMed Central

2011-01-01

Background Inflammatory bowel disease (IBD) is thought to be due to an abnormal interaction between the host immune system and commensal microflora. Within the intestinal immune system, B cells produce physiologically natural antibodies but pathologically atypical anti-neutrophil antibodies (xANCAs) are frequently observed in patients with IBD. The objective is to investigate the localisation of immunoglobulin-producing cells (IPCs) in samples of inflamed intestinal tissue taken from patients with IBD, and their possible relationship with clinical features. Methods The IPCs in small intestinal, colonic and rectal biopsy specimens of patients with IBD were analysed by means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The B cell phenotype of the IPC-positive samples was assessed using monoclonal antibodies specific for CD79, CD20, CD23, CD21, CD5, λ and κ chains. Statistical correlations were sought between the histological findings and clinical expression. Results The study involved 96 patients (64 with ulcerative colitis and 32 with Crohn's disease). Two different patterns of B lymphocyte infiltrates were found in the intestinal tissue: one was characterised by a strong to moderate stromal localisation of small IgM+/CD79+/CD20-/CD21-/CD23-/CD5± IPCs (42.7% of cases); in the other (57.3%) no such small IPCs were detected in stromal or epithelial tissues. IPCs were significantly less frequent in the patients with Crohn's disease than in those with ulcerative colitis (p = 0.004). Conclusion Our findings suggest that different immunopathogenetic pathways underlie chronic intestinal inflammation with different clinical expressions. The presence of small B lymphocytes resembling B-1 cells also seemed to be negatively associated with Crohn's disease. It can therefore be inferred that the gut contains an alternative population of B cells that have a regulatory function. PMID:22208453

Derivation of novel human ground state naive pluripotent stem cells.

PubMed

Gafni, Ohad; Weinberger, Leehee; Mansour, Abed AlFatah; Manor, Yair S; Chomsky, Elad; Ben-Yosef, Dalit; Kalma, Yael; Viukov, Sergey; Maza, Itay; Zviran, Asaf; Rais, Yoach; Shipony, Zohar; Mukamel, Zohar; Krupalnik, Vladislav; Zerbib, Mirie; Geula, Shay; Caspi, Inbal; Schneir, Dan; Shwartz, Tamar; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Benjamin, Sima; Amit, Ido; Tanay, Amos; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2013-12-12

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

In Vitro Tissue-Engineered Skeletal Muscle Models for Studying Muscle Physiology and Disease.

PubMed

Khodabukus, Alastair; Prabhu, Neel; Wang, Jason; Bursac, Nenad

2018-04-25

Healthy skeletal muscle possesses the extraordinary ability to regenerate in response to small-scale injuries; however, this self-repair capacity becomes overwhelmed with aging, genetic myopathies, and large muscle loss. The failure of small animal models to accurately replicate human muscle disease, injury and to predict clinically-relevant drug responses has driven the development of high fidelity in vitro skeletal muscle models. Herein, the progress made and challenges ahead in engineering biomimetic human skeletal muscle tissues that can recapitulate muscle development, genetic diseases, regeneration, and drug response is discussed. Bioengineering approaches used to improve engineered muscle structure and function as well as the functionality of satellite cells to allow modeling muscle regeneration in vitro are also highlighted. Next, a historical overview on the generation of skeletal muscle cells and tissues from human pluripotent stem cells, and a discussion on the potential of these approaches to model and treat genetic diseases such as Duchenne muscular dystrophy, is provided. Finally, the need to integrate multiorgan microphysiological systems to generate improved drug discovery technologies with the potential to complement or supersede current preclinical animal models of muscle disease is described. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intestinal epithelial wound healing assay in an epithelial-mesenchymal co-culture system.

PubMed

Seltana, Amira; Basora, Nuria; Beaulieu, Jean-François

2010-01-01

Rapid and efficient healing of epithelial damage is critical to the functional integrity of the small intestine. Epithelial repair is a complex process that has largely been studied in cultured epithelium but to a much lesser extent in mucosa. We describe a novel method for the study of wound healing using a co-culture system that combined an intestinal epithelial Caco-2/15 cell monolayer cultured on top of human intestinal myofibroblasts, which together formed a basement membrane-like structure that contained many of the major components found at the epithelial-mesenchymal interface in the human intestine. To investigate the mechanism of restitution, small lesions were generated in epithelial cell monolayers on plastic or in co-cultures without disturbing the underlying mesenchymal layer. Monitoring of wound healing showed that repair was more efficient in Caco-2/15-myofibroblast co-cultures than in Caco-2/15 monolayers and involved the deposition of basement membrane components. Functional experiments showed that the addition of type I collagen or human fibronectin to the culture medium significantly accelerated wound closure on epithelial cell co-cultures. This system may provide a new tool to investigate the mechanisms that regulate wound healing in the intestinal epithelium.

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction

PubMed Central

Zhou, Jian; Alvarez-Elizondo, Martha B.; Botvinick, Elliot

2012-01-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca2+ wave in the epithelium, and multiple Ca2+ waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca2+ or decreasing intracellular Ca2+ both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca2+-dependent smooth muscle shortening. PMID:22114176

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction.

PubMed

Zhou, Jian; Alvarez-Elizondo, Martha B; Botvinick, Elliot; George, Steven C

2012-02-01

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-μm diameter) in rat lung slices (∼250-μm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.

Attenuation of the DNA Damage Response by Transforming Growth Factor-Beta Inhibitors Enhances Radiation Sensitivity of Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo

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

Du, Shisuo; Bouquet, Sophie; Lo, Chen-Hao

2015-01-01

Purpose: To determine whether transforming growth factor (TGF)-β inhibition increases the response to radiation therapy in human and mouse non–small-cell lung carcinoma (NSCLC) cells in vitro and in vivo. Methods and Materials: TGF-β–mediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-β type 1 receptor kinase, or with the pan-isoform TGF-β neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, γH2AX foci formation,more » or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody. Results: The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced γH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-β signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-β neutralizing antibody exhibited fewer γH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation. Conclusions: Inhibition of TGF-β before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-β inhibition and RT to provide therapeutic benefit in NSCLC.« less

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells.

PubMed

Hoffman, Joseph F; Joiner, William; Nehrke, Keith; Potapova, Olga; Foye, Kristen; Wickrema, Amittha

2003-06-10

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external Ko+ and remains refractory to Ko+ added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside-out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37 degrees C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside-out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.

Chlorella vulgaris Induces Apoptosis of Human Non-Small Cell Lung Carcinoma (NSCLC) Cells.

PubMed

Zhang, Zhi-Dong; Liang, Kai; Li, Kun; Wang, Guo-Quan; Zhang, Ke-Wei; Cai, Lei; Zhai, Shui-Ting; Chou, Kuo-Chen

2017-01-01

Chlorella vulgaris (C. vulgaris), a unicellular green microalga, has been widely used as a food supplement and reported to have antioxidant and anticancer properties. The current study was designed to assess the cytotoxic, apoptotic, and DNA-damaging effects of C. vulgaris growth factor (CGF), hot water C. vulgaris extracts, inlung tumor A549 and NCI-H460 cell lines. A549 cells, NCI-H460 cells, and normal human fibroblasts were treated with CGF at various concentrations (0-300 μg/ml) for 24 hr. The comet assay and γH2AX assay showed DNA damage in A549 and NCI-H460 cells upon CGF exposure. Evaluation of apoptosis by the TUNEL assay and DNA fragmentation analysis by agarose gel electrophoresis showed that CGF induced apoptosis in A549 and NCI-H460 cells. Chlorella vulgaris hot water extract induced apoptosis and DNA damage in human lung carcinoma cells. CGF can thus be considered a potential cytotoxic or genotoxic drug for treatment of lung carcinoma. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

MicroRNA-944 Affects Cell Growth by Targeting EPHA7 in Non-Small Cell Lung Cancer.

PubMed

Liu, Minxia; Zhou, Kecheng; Cao, Yi

2016-09-26

MicroRNAs (miRNAs) have critical roles in lung tumorigenesis and development. To determine aberrantly expressed miRNAs involved in non-small cell lung cancer (NSCLC) and investigate pathophysiological functions and mechanisms, we firstly carried out small RNA deep sequencing in NSCLC cell lines (EPLC-32M1, A549 and 801D) and a human immortalized cell line 16HBE, we then studied miRNA function by cell proliferation and apoptosis. cDNA microarray, luciferase reporter assay and miRNA transfection were used to investigate interaction between the miRNA and target gene. miR-944 was significantly down-regulated in NSCLC and had many putative targets. Moreover, the forced expression of miR-944 significantly inhibited the proliferation of NSCLC cells in vitro. By integrating mRNA expression data and miR-944-target prediction, we disclosed that EPHA7 was a potential target of miR-944, which was further verified by luciferase reporter assay and microRNA transfection. Our data indicated that miR-944 targets EPHA7 in NSCLC and regulates NSCLC cell proliferation, which may offer a new mechanism underlying the development and progression of NSCLC.

Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity

PubMed Central

Ratajczak, Mariusz Z.; Zuba-Surma, Ewa K.; Shin, Dong-Myung; Ratajczak, Janina; Kucia, Magda

2011-01-01

Recently, we purified rare CXC chemokine receptor 4 expressing (CXCR4+) small stem cells (SCs) from the murine bone marrow (BM) that express markers characteristic for embryonic (E)SCs, epiblast (EP)SCs, and primordial germ cells (PGCs). We named these primitive cells very small embryonic-like (VSEL) SCs (VSELs). Our data indicate that VSELs are also present in many other organs in mice and that they may differentiate into cells from all three germ layers. Similar SCs were also isolated from human cord blood (CB) and mobilized peripheral blood (mPB). We hypothesize that VSELs are deposited during gastrulation and organogenesis in developing organs/tissues of mammals as a population of pluripotent stem cells (PSCs) that give rise to tissue committed monopotent SCs and that their number decreases with age. Therefore VSELs could play a pivotal role in normal rejuvenation of adult tissues as well as involvement in regeneration of damaged organs. Thus, these cells are potential SCs candidates for regenerative medicine and we envision that the regenerative potential of these cells could be harnessed to decelerate the aging processes. PMID:18601995

Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

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

Rong-Nan Huang; I-Ching Ho; Ling-Hui Yih

Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 {mu}M) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplicationmore » in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. 61 refs., 6 figs., 2 tabs.« less

Discovery of a Positive Allosteric Modulator of the Thyrotropin Receptor: Potentiation of Thyrotropin-Mediated Preosteoblast Differentiation In Vitro

PubMed Central

Eliseeva, Elena; Boutin, Alisa; Barnaeva, Elena; Ferrer, Marc; Southall, Noel; Kim, David; Hu, Xin; Morgan, Sarah J.; Marugan, Juan J.; Gershengorn, Marvin C.

2018-01-01

Recently, we showed that TSH-enhanced differentiation of a human preosteoblast-like cell model involved a β-arrestin 1 (β-Arr 1)-mediated pathway. To study this pathway in more detail, we sought to discover a small molecule ligand that was functionally selective toward human TSH receptor (TSHR) activation of β-Arr 1. High-throughput screening using a cell line stably expressing mutated TSHRs and mutated β-Arr 1 (DiscoverX1 cells) led to the discovery of agonists that stimulated translocation of β-Arr 1 to the TSHR, but did not activate Gs-mediated signaling pathways, i.e., cAMP production. D3-βArr (NCGC00379308) was selected. In DiscoverX1 cells, D3-βArr stimulated β-Arr 1 translocation with a 5.1-fold greater efficacy than TSH and therefore potentiated the effect of TSH in stimulating β-Arr 1 translocation. In human U2OS-TSHR cells expressing wild-type TSHRs, which is a model of human preosteoblast-like cells, TSH upregulated the osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). D3-βArr alone had only a weak effect to upregulate these bone markers, but D3-βArr potentiated TSH-induced upregulation of ALPL and OPN mRNA levels 1.6-fold and 5.5-fold, respectively, at the maximum dose of ligands. Furthermore, the positive allosteric modulator effect of D3-βArr resulted in an increase of TSH-induced secretion of OPN protein. In summary, we have discovered the first small molecule positive allosteric modulator of TSHR. As D3-βArr potentiates the effect of TSH to enhance differentiation of a human preosteoblast in an in vitro model, it will allow a novel experimental approach for probing the role of TSH-induced β-Arr 1 signaling in osteoblast differentiation. PMID:29089368

Discovery of a Positive Allosteric Modulator of the Thyrotropin Receptor: Potentiation of Thyrotropin-Mediated Preosteoblast Differentiation In Vitro.

PubMed

Neumann, Susanne; Eliseeva, Elena; Boutin, Alisa; Barnaeva, Elena; Ferrer, Marc; Southall, Noel; Kim, David; Hu, Xin; Morgan, Sarah J; Marugan, Juan J; Gershengorn, Marvin C

2018-01-01

Recently, we showed that TSH-enhanced differentiation of a human preosteoblast-like cell model involved a β -arrestin 1 ( β -Arr 1)-mediated pathway. To study this pathway in more detail, we sought to discover a small molecule ligand that was functionally selective toward human TSH receptor (TSHR) activation of β -Arr 1. High-throughput screening using a cell line stably expressing mutated TSHRs and mutated β -Arr 1 (DiscoverX1 cells) led to the discovery of agonists that stimulated translocation of β -Arr 1 to the TSHR, but did not activate G s -mediated signaling pathways, i.e., cAMP production. D3- β Arr (NCGC00379308) was selected. In DiscoverX1 cells, D3- β Arr stimulated β -Arr 1 translocation with a 5.1-fold greater efficacy than TSH and therefore potentiated the effect of TSH in stimulating β -Arr 1 translocation. In human U2OS-TSHR cells expressing wild-type TSHRs, which is a model of human preosteoblast-like cells, TSH upregulated the osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). D3- β Arr alone had only a weak effect to upregulate these bone markers, but D3- β Arr potentiated TSH-induced upregulation of ALPL and OPN mRNA levels 1.6-fold and 5.5-fold, respectively, at the maximum dose of ligands. Furthermore, the positive allosteric modulator effect of D3- β Arr resulted in an increase of TSH-induced secretion of OPN protein. In summary, we have discovered the first small molecule positive allosteric modulator of TSHR. As D3- β Arr potentiates the effect of TSH to enhance differentiation of a human preosteoblast in an in vitro model, it will allow a novel experimental approach for probing the role of TSH-induced β -Arr 1 signaling in osteoblast differentiation. U.S. Government work not protected by U.S. copyright.

BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

PubMed

Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

2015-11-01

Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

Tangeretin inhibits extracellular-signal-regulated kinase (ERK) phosphorylation.

PubMed

Van Slambrouck, Séverine; Parmar, Virinder S; Sharma, Sunil K; De Bondt, Bart; Foré, Fleur; Coopman, Peter; Vanhoecke, Barbara W; Boterberg, Tom; Depypere, Herman T; Leclercq, Guy; Bracke, Marc E

2005-03-14

Tangeretin is a methoxyflavone from citrus fruits, which inhibits growth of human mammary cancer cells and cytolysis by natural killer cells. Attempting to unravel the flavonoid's action mechanism, we found that it inhibited extracellular-signal-regulated kinases 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent way. In human T47D mammary cancer cells this inhibition was optimally observed after priming with estradiol. The spectrum of the intracellular signalling kinase inhibition was narrow and comparison of structural congeners showed that inhibition of ERK phosphorylation was not unique for tangeretin. Our data add tangeretin to the list of small kinase inhibitors with a restricted intracellular inhibition profile.

Multiparametric Phenotypic Screening System for Profiling Bioactive Compounds Using Human Fetal Hippocampal Neural Stem/Progenitor Cells.

PubMed

Tabata, Yoshikuni; Murai, Norio; Sasaki, Takeo; Taniguchi, Sachie; Suzuki, Shuichi; Yamazaki, Kazuto; Ito, Masashi

2015-10-01

Stem cell research has been progressing rapidly, contributing to regenerative biology and regenerative medicine. In this field, small-molecule compounds affecting stem cell proliferation/differentiation have been explored to understand stem cell biology and support regenerative medicine. In this study, we established a multiparametric screening system to detect bioactive compounds affecting the cell fate of human neural stem/progenitor cells (NSCs/NPCs), using human fetal hippocampal NSCs/NPCs, HIP-009 cells. We examined effects of 410 compounds, which were collected based on mechanisms of action (MOAs) and chemotypes, on HIP-009's cell fate (self-renewal, neuronal and astrocytic differentiation) and morphology by automated multiparametric assays and profiled induced cellular phenotypes. We found that this screening classified compounds with the same MOAs into subgroups according to additional pharmacological effects (e.g., mammalian target of rapamycin complex 1 [mTORC1] inhibitors and mTORC1/mTORC2 dual inhibitors among mTOR inhibitors). Moreover, it identified compounds that have off-target effects under matrix analyses of MOAs and structure similarities (e.g., neurotropic effects of amitriptyline among tri- and tetracyclic compounds). Therefore, this automated, medium-throughput and multiparametric screening system is useful for finding compounds that affect the cell fate of human NSCs/NPCs for supporting regenerative medicine and to fingerprint compounds based on human stem cells' multipotency, leading to understanding of stem cell biology. © 2015 Society for Laboratory Automation and Screening.

Increased Cardiac Myocyte Progenitors in Failing Human Hearts

PubMed Central

Kubo, Hajime; Jaleel, Naser; Kumarapeli, Asangi; Berretta, Remus M.; Bratinov, George; Shan, Xiaoyin; Wang, Hongmei; Houser, Steven R.; Margulies, Kenneth B.

2009-01-01

Background Increasing evidence, derived mainly from animal models, supports the existence of endogenous cardiac renewal and repair mechanisms in adult mammalian hearts that could contribute to normal homeostasis and the responses to pathological insults. Methods and Results Translating these results, we isolated small c-kit+ cells from 36 of 37 human hearts using primary cell isolation techniques and magnetic cell sorting techniques. The abundance of these cardiac progenitor cells was increased nearly 4-fold in patients with heart failure requiring transplantation compared with nonfailing controls. Polychromatic flow cytometry of primary cell isolates (<30 μm) without antecedent c-kit enrichment confirmed the increased abundance of c-kit+ cells in failing hearts and demonstrated frequent coexpression of CD45 in these cells. Immunocytochemical characterization of freshly isolated, c-kit–enriched human cardiac progenitor cells confirmed frequent coexpression of c-kit and CD45. Primary cardiac progenitor cells formed new human cardiac myocytes at a relatively high frequency after coculture with neonatal rat ventricular myocytes. These contracting new cardiac myocytes exhibited an immature phenotype and frequent electric coupling with the rat myocytes that induced their myogenic differentiation. Conclusions Despite the increased abundance and cardiac myogenic capacity of cardiac progenitor cells in failing human hearts, the need to replace these organs via transplantation implies that adverse features of the local myocardial environment overwhelm endogenous cardiac repair capacity. Developing strategies to improve the success of endogenous cardiac regenerative processes may permit therapeutic myocardial repair without cell delivery per se. PMID:18645055

Glycyrrhetinic acid induces G1-phase cell cycle arrest in human non-small cell lung cancer cells through endoplasmic reticulum stress pathway

PubMed Central

ZHU, JIE; CHEN, MEIJUAN; CHEN, NING; MA, AIZHEN; ZHU, CHUNYAN; ZHAO, RUOLIN; JIANG, MIAO; ZHOU, JING; YE, LIHONG; FU, HAIAN; ZHANG, XU

2015-01-01

Glycyrrhetinic acid (GA) is a natural compound extracted from liquorice, which is often used in traditional Chinese medicine. The purpose of the present study was to investigate the antitumor effect of GA in human non-small cell lung cancer (NSCLC), and its underlying mechanisms in vitro. We have shown that GA suppressed the proliferation of A549 and NCI-H460 cells. Flow cytometric analysis showed that GA arrested cell cycle in G0/G1 phase without inducing apoptosis. Western blot analysis indicated that GA mediated G1-phase cell cycle arrest by upregulation of cyclin-dependent kinase inhibitors (CKIs) (p18, p16, p27 and p21) and inhibition of cyclins (cyclin-D1, -D3 and -E) and cyclin-dependent kinases (CDKs) (CDK4, 6 and 2). GA also maintained pRb phosphorylation status, and inhibited E2F transcription factor 1 (E2F-1) in both cell lines. GA upregulated the unfolded proteins, Bip, PERK and ERP72. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggered the unfolded protein response (UPR), which could be the mechanism by which GA inhibited cell proliferation in NSCLC cells. GA then coordinated the induction of ER chaperones, which decreased protein synthesis and induced cell cycle arrest in the G1 phase. This study provides experimental evidence to support the development of GA as a chemotherapeutic agent for NSCLC. PMID:25573651

Bag3 promotes resistance to apoptosis through Bcl-2 family members in non-small cell lung cancer.

PubMed

Zhang, Yong; Wang, Jian-Hua; Lu, Qiang; Wang, Yun-Jie

2012-01-01

In non-small cell lung cancer (NSCLC) certain molecular characteristics, which are related to molecular alterations have been investigated. These are responsible for both the initiation and maintenance of the malignancy in lung cancer. The aim of this study was to evaluate the influence of Bag3 (Bcl-2 associated athanogene 3) in the regulation of apoptosis on NSCLC. Bag3 and Hsp70 expression were examined by immunohistochemistry to confirm their potential roles in the prevalence of NSCLC. We also established human normal bronchial epithelial cells and HOP-62 cell line as the model to analyze cell apoptosis and the expression of Hsp70, Bcl-XL and Bcl-2, which were affected by Bag3. In this study, we found that Bag3 and Hsp70 are highly expressed in few tissues and cell lines of NSCLC. Bag3 inhibits apoptosis in human normal bronchial epithelial cell lines and sustain the survival of NSCLC cells. Bag3, Hsp70, Bcl-XL and Bcl-2 are up-regulated in NSCLC cell lines. At the same time, the silencing of Bag3 results in diminishing protein levels of Bcl-XL and Bcl-2. The results of immunoprecipitation identified that Bag3 could interact with Hsp70, Bcl-XL and Bcl-2 NSCLC cells directly or indirectly. We conclude that NSCLC cells were protected from apoptosis through increasing Bag3 expression and consequently promoted the expression of Bcl-XL and Bcl-2.

Classification of human natural killer cells based on migration behavior and cytotoxic response.

PubMed

Vanherberghen, Bruno; Olofsson, Per E; Forslund, Elin; Sternberg-Simon, Michal; Khorshidi, Mohammad Ali; Pacouret, Simon; Guldevall, Karolin; Enqvist, Monika; Malmberg, Karl-Johan; Mehr, Ramit; Önfelt, Björn

2013-02-21

Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.

New Small Molecules Targeting Apoptosis and Cell Viability in Osteosarcoma

PubMed Central

Maugg, Doris; Rothenaigner, Ina; Schorpp, Kenji; Potukuchi, Harish Kumar; Korsching, Eberhard; Baumhoer, Daniel; Hadian, Kamyar

2015-01-01

Despite the option of multimodal therapy in the treatment strategies of osteosarcoma (OS), the most common primary malignant bone tumor, the standard therapy has not changed over the last decades and still involves multidrug chemotherapy and radical surgery. Although successfully applied in many patients a large number of patients eventually develop recurrent or metastatic disease in which current therapeutic regimens often lack efficacy. Thus, new therapeutic strategies are urgently needed. In this study, we performed a phenotypic high-throughput screening campaign using a 25,000 small-molecule diversity library to identify new small molecules selectively targeting osteosarcoma cells. We could identify two new small molecules that specifically reduced cell viability in OS cell lines U2OS and HOS, but affected neither hepatocellular carcinoma cell line (HepG2) nor primary human osteoblasts (hOB). In addition, the two compounds induced caspase 3 and 7 activity in the U2OS cell line. Compared to conventional drugs generally used in OS treatment such as doxorubicin, we indeed observed a greater sensitivity of OS cell viability to the newly identified compounds compared to doxorubicin and staurosporine. The p53-negative OS cell line Saos-2 almost completely lacked sensitivity to compound treatment that could indicate a role of p53 in the drug response. Taken together, our data show potential implications for designing more efficient therapies in OS. PMID:26039064

A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

PubMed

Ratajczak, Mariusz Z; Ratajczak, Janina; Suszynska, Malwina; Miller, Donald M; Kucia, Magda; Shin, Dong-Myung

2017-01-06

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine. © 2017 American Heart Association, Inc.

Genetic engineering of human embryonic stem cells with lentiviral vectors.

PubMed

Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

2005-08-01

Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

Carfilzomib demonstrates broad anti-tumor activity in pre-clinical non-small cell and small cell lung cancer models.

PubMed

Baker, Amanda F; Hanke, Neale T; Sands, Barbara J; Carbajal, Liliana; Anderl, Janet L; Garland, Linda L

2014-12-31

Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1B studies of CFZ reported signals of clinical activity in solid tumors, including small cell lung cancer (SCLC). The aim of this study was to investigate the activity of CFZ in lung cancer models. A diverse panel of human lung cancer cell lines and a SHP77 small cell lung cancer xenograft model were used to investigate the anti-tumor activity of CFZ. CFZ treatment inhibited both the constitutive proteasome and the immunoproteasome in lung cancer cell lines. CFZ had marked anti-proliferative activity in A549, H1993, H520, H460, and H1299 non-small cell lung cancer (NSCLC) cell lines, with IC50 values after 96 hour exposure from <1.0 nM to 36 nM. CFZ had more variable effects in the SHP77 and DMS114 SCLC cell lines, with IC50 values at 96 hours from <1 nM to 203 nM. Western blot analysis of CFZ-treated H1993 and SHP77 cells showed cleavage of poly ADP ribose polymerase (PARP) and caspase-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. In SHP77 flank xenograft tumors, CFZ monotherapy inhibited tumor growth and prolonged survival, while no additive or synergistic anti-tumor efficacy was observed for CFZ + cisplatin (CDDP). CFZ demonstrated anti-proliferative activity in lung cancer cell lines in vitro and resulted in a significant survival advantage in mice with SHP77 SCLC xenografts, supporting further pre-clinical and clinical investigations of CFZ in NSCLC and SCLC.

Gamma-aminobutyric acid, a potential tumor suppressor for small airway-derived lung adenocarcinoma.

PubMed

Schuller, Hildegard M; Al-Wadei, Hussein A N; Majidi, Mourad

2008-10-01

Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer in smokers and non-smokers that arises in most cases from small airway epithelial cells. PAC has a high mortality due to its aggressive behavior and resistance to cancer therapeutics. We have shown previously that the proliferation of human PAC cells NCI-H322 and immortalized human small airway epithelial cells HPL1D is stimulated by cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent phosphorylation of cyclic adenosine monophosphate response element-binding (CREB) protein and transactivation of the epidermal growth factor receptor and that this pathway is activated by beta-1-adrenoreceptors (beta(1)-ARs) and the non-genomic estrogen receptor beta. Our current in vitro studies with HPL1D and NCI-H322 cells showed that signaling via the gamma-amino butyric acid receptor (GABA(B)R) strongly inhibited base level and isoproterenol-induced cAMP, p-CREB, cyclic adenosine monophosphate response element-luciferase activity and p-extracellular regulated kinase-1 (ERK1)/2 and effectively blocked DNA synthesis and cell migration. The inhibitory effects of gamma-amino butyric acid (GABA) were disinhibited by the GABA(B)R antagonist CGP-35348 or GABA(B)R knockdown. Immunohistochemical investigation of hamster lungs showed significant underexpression of GABA in animals with small airway-derived PACs induced by the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These findings suggest that GABA may have tumor suppressor function in small airway epithelia and the PACs derived from them and that downregulation of GABA by NNK may contribute to the development of this cancer in smokers. Our findings suggest that marker-guided treatment with GABA or a GABA(B)R agonist of individuals with downregulated pulmonary GABA may provide a novel targeted approach for the prevention of PAC in smokers.

New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III.

PubMed

Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

2014-01-01

Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells.

Addressable droplet microarrays for single cell protein analysis.

PubMed

Salehi-Reyhani, Ali; Burgin, Edward; Ces, Oscar; Willison, Keith R; Klug, David R

2014-11-07

Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

Development of an on-line exposure system to determine freshly produced diesel engine emission-induced cellular effects.

PubMed

Oostingh, Gertie J; Papaioannou, Eleni; Chasapidis, Leonidas; Akritidis, Theofylaktos; Konstandopoulos, Athanasios G; Duschl, Albert

2013-09-01

Diesel engine emission particle filters are often placed at exhaust outlets to remove particles from the exhaust. The use of filters results in the exposure to a reduced number of nanometer-sized particles, which might be more harmful than the exposure to a larger number of micrometer-sized particles. An in vitro exposure system was established to expose human alveolar epithelial cells to freshly generated exhaust. Computer simulations were used to determine the optimal flow characteristics and ensure equal exposure conditions for each well of a 6-well plate. A selective particle size sampler was used to continuously deliver diesel soot particles with different particle size distributions to cells in culture. To determine, whether the system could be used for cellular assays, alterations in cytokine production and cell viability of human alveolar A549 cells were determined after 3h on-line exposure followed by a 21-h conventional incubation period. Data indicated that complete diesel engine emission slightly affected pre-stimulated cells, but naive cells were not affected. The fractions containing large or small particles never affected the cells. The experimental set-up allowed a reliable exposure of the cells to the complete exhaust fraction or to the fractions containing either large or small diesel engine emission particles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Highly efficient radiosensitization of human glioblastoma and lung cancer cells by a G-quadruplex DNA binding compound.

PubMed

Merle, Patrick; Gueugneau, Marine; Teulade-Fichou, Marie-Paule; Müller-Barthélémy, Mélanie; Amiard, Simon; Chautard, Emmanuel; Guetta, Corinne; Dedieu, Véronique; Communal, Yves; Mergny, Jean-Louis; Gallego, Maria; White, Charles; Verrelle, Pierre; Tchirkov, Andreï

2015-11-06

Telomeres are nucleoprotein structures at the end of chromosomes which stabilize and protect them from nucleotidic degradation and end-to-end fusions. The G-rich telomeric single-stranded DNA overhang can adopt a four-stranded G-quadruplex DNA structure (G4). Stabilization of the G4 structure by binding of small molecule ligands enhances radiosensitivity of tumor cells, and this combined treatment represents a novel anticancer approach. We studied the effect of the platinum-derived G4-ligand, Pt-ctpy, in association with radiation on human glioblastoma (SF763 and SF767) and non-small cell lung cancer (A549 and H1299) cells in vitro and in vivo. Treatments with submicromolar concentrations of Pt-ctpy inhibited tumor proliferation in vitro with cell cycle alterations and induction of apoptosis. Non-toxic concentrations of the ligand were then combined with ionizing radiation. Pt-ctpy radiosensitized all cell lines with dose-enhancement factors between 1.32 and 1.77. The combined treatment led to increased DNA breaks. Furthermore, a significant radiosensitizing effect of Pt-ctpy in mice xenografted with glioblastoma SF763 cells was shown by delayed tumor growth and improved survival. Pt-ctpy can act in synergy with radiation for efficient killing of cancer cells at concentrations at which it has no obvious toxicity per se, opening perspectives for future therapeutic applications.

L1000CDS2: LINCS L1000 characteristic direction signatures search engine

PubMed Central

Duan, Qiaonan; Reid, St Patrick; Clark, Neil R; Wang, Zichen; Fernandez, Nicolas F; Rouillard, Andrew D; Readhead, Ben; Tritsch, Sarah R; Hodos, Rachel; Hafner, Marc; Niepel, Mario; Sorger, Peter K; Dudley, Joel T; Bavari, Sina; Panchal, Rekha G; Ma’ayan, Avi

2016-01-01

The library of integrated network-based cellular signatures (LINCS) L1000 data set currently comprises of over a million gene expression profiles of chemically perturbed human cell lines. Through unique several intrinsic and extrinsic benchmarking schemes, we demonstrate that processing the L1000 data with the characteristic direction (CD) method significantly improves signal to noise compared with the MODZ method currently used to compute L1000 signatures. The CD processed L1000 signatures are served through a state-of-the-art web-based search engine application called L1000CDS2. The L1000CDS2 search engine provides prioritization of thousands of small-molecule signatures, and their pairwise combinations, predicted to either mimic or reverse an input gene expression signature using two methods. The L1000CDS2 search engine also predicts drug targets for all the small molecules profiled by the L1000 assay that we processed. Targets are predicted by computing the cosine similarity between the L1000 small-molecule signatures and a large collection of signatures extracted from the gene expression omnibus (GEO) for single-gene perturbations in mammalian cells. We applied L1000CDS2 to prioritize small molecules that are predicted to reverse expression in 670 disease signatures also extracted from GEO, and prioritized small molecules that can mimic expression of 22 endogenous ligand signatures profiled by the L1000 assay. As a case study, to further demonstrate the utility of L1000CDS2, we collected expression signatures from human cells infected with Ebola virus at 30, 60 and 120 min. Querying these signatures with L1000CDS2 we identified kenpaullone, a GSK3B/CDK2 inhibitor that we show, in subsequent experiments, has a dose-dependent efficacy in inhibiting Ebola infection in vitro without causing cellular toxicity in human cell lines. In summary, the L1000CDS2 tool can be applied in many biological and biomedical settings, while improving the extraction of knowledge from the LINCS L1000 resource. PMID:28413689

Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease.

PubMed

Dinca, Ana; Chien, Wei-Ming; Chin, Michael T

2016-02-22

Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

A three-dimensional model of human lung development and disease from pluripotent stem cells.

PubMed

Chen, Ya-Wen; Huang, Sarah Xuelian; de Carvalho, Ana Luisa Rodrigues Toste; Ho, Siu-Hong; Islam, Mohammad Naimul; Volpi, Stefano; Notarangelo, Luigi D; Ciancanelli, Michael; Casanova, Jean-Laurent; Bhattacharya, Jahar; Liang, Alice F; Palermo, Laura M; Porotto, Matteo; Moscona, Anne; Snoeck, Hans-Willem

2017-05-01

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modelling, drug discovery and regenerative medicine. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.

Rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase C epsilon.

PubMed

Filone, Claire Marie; Hanna, Sheri L; Caino, M Cecilia; Bambina, Shelly; Doms, Robert W; Cherry, Sara

2010-11-24

As an arthropod-borne human pathogen, Rift Valley fever virus (RVFV) cycles between an insect vector and mammalian hosts. Little is known about the cellular requirements for infection in either host. Here we developed a tissue culture model for RVFV infection of human and insect cells that is amenable to high-throughput screening. Using this approach we screened a library of 1280 small molecules with pharmacologically defined activities and identified 59 drugs that inhibited RVFV infection with 15 inhibiting RVFV replication in both human and insect cells. Amongst the 15 inhibitors that blocked infection in both hosts was a subset that inhibits protein kinase C. Further studies found that infection is dependent upon the novel protein kinase C isozyme epsilon (PKCε) in both human and insect cells as well as in adult flies. Altogether, these data show that inhibition of cellular factors required for early steps in the infection cycle including PKCε can block RVFV infection, and may represent a starting point for the development of anti-RVFV therapeutics.

Tissue-specific mutation accumulation in human adult stem cells during life

NASA Astrophysics Data System (ADS)

Blokzijl, Francis; de Ligt, Joep; Jager, Myrthe; Sasselli, Valentina; Roerink, Sophie; Sasaki, Nobuo; Huch, Meritxell; Boymans, Sander; Kuijk, Ewart; Prins, Pjotr; Nijman, Isaac J.; Martincorena, Inigo; Mokry, Michal; Wiegerinck, Caroline L.; Middendorp, Sabine; Sato, Toshiro; Schwank, Gerald; Nieuwenhuis, Edward E. S.; Verstegen, Monique M. A.; van der Laan, Luc J. W.; de Jonge, Jeroen; Ijzermans, Jan N. M.; Vries, Robert G.; van de Wetering, Marc; Stratton, Michael R.; Clevers, Hans; Cuppen, Edwin; van Boxtel, Ruben

2016-10-01

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

Anatomically realistic multiscale models of normal and abnormal gastrointestinal electrical activity

PubMed Central

Cheng, Leo K; Komuro, Rie; Austin, Travis M; Buist, Martin L; Pullan, Andrew J

2007-01-01

One of the major aims of the International Union of Physiological Sciences (IUPS) Physiome Project is to develop multiscale mathematical and computer models that can be used to help understand human health. We present here a small facet of this broad plan that applies to the gastrointestinal system. Specifically, we present an anatomically and physiologically based modelling framework that is capable of simulating normal and pathological electrical activity within the stomach and small intestine. The continuum models used within this framework have been created using anatomical information derived from common medical imaging modalities and data from the Visible Human Project. These models explicitly incorporate the various smooth muscle layers and networks of interstitial cells of Cajal (ICC) that are known to exist within the walls of the stomach and small bowel. Electrical activity within individual ICCs and smooth muscle cells is simulated using a previously published simplified representation of the cell level electrical activity. This simulated cell level activity is incorporated into a bidomain representation of the tissue, allowing electrical activity of the entire stomach or intestine to be simulated in the anatomically derived models. This electrical modelling framework successfully replicates many of the qualitative features of the slow wave activity within the stomach and intestine and has also been used to investigate activity associated with functional uncoupling of the stomach. PMID:17457969

Identifying a Small Molecule Blocking Antigen Presentation in Autoimmune Thyroiditis.

PubMed

Li, Cheuk Wun; Menconi, Francesca; Osman, Roman; Mezei, Mihaly; Jacobson, Eric M; Concepcion, Erlinda; David, Chella S; Kastrinsky, David B; Ohlmeyer, Michael; Tomer, Yaron

2016-02-19

We previously showed that an HLA-DR variant containing arginine at position 74 of the DRβ1 chain (DRβ1-Arg74) is the specific HLA class II variant conferring risk for autoimmune thyroid diseases (AITD). We also identified 5 thyroglobulin (Tg) peptides that bound to DRβ1-Arg74. We hypothesized that blocking the binding of these peptides to DRβ1-Arg74 could block the continuous T-cell activation in thyroiditis needed to maintain the autoimmune response to the thyroid. The aim of the current study was to identify small molecules that can block T-cell activation by Tg peptides presented within DRβ1-Arg74 pockets. We screened a large and diverse library of compounds and identified one compound, cepharanthine that was able to block peptide binding to DRβ1-Arg74. We then showed that Tg.2098 is the dominant peptide when inducing experimental autoimmune thyroiditis (EAT) in NOD mice expressing human DRβ1-Arg74. Furthermore, cepharanthine blocked T-cell activation by thyroglobulin peptides, in particular Tg.2098 in mice that were induced with EAT. For the first time we identified a small molecule that can block Tg peptide binding and presentation to T-cells in autoimmune thyroiditis. If confirmed cepharanthine could potentially have a role in treating human AITD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The significance of PIWI family expression in human lung embryogenesis and non-small cell lung cancer.

PubMed

Navarro, Alfons; Tejero, Rut; Viñolas, Nuria; Cordeiro, Anna; Marrades, Ramon M; Fuster, Dolors; Caritg, Oriol; Moises, Jorge; Muñoz, Carmen; Molins, Laureano; Ramirez, Josep; Monzo, Mariano

2015-10-13

The expression of Piwi-interacting RNAs, small RNAs that bind to PIWI proteins, was until recently believed to be limited to germinal stem cells. We have studied the expression of PIWI genes during human lung embryogenesis and in paired tumor and normal tissue prospectively collected from 71 resected non-small-cell lung cancer patients. The mRNA expression analysis showed that PIWIL1 was highly expressed in 7-week embryos and downregulated during the subsequent weeks of development. PIWIL1 was expressed in 11 of the tumor samples but in none of the normal tissue samples. These results were validated by immunohistochemistry, showing faint cytoplasmic reactivity in the PIWIL1-positive samples. Interestingly, the patients expressing PIWIL1 had a shorter time to relapse (TTR) (p = 0.006) and overall survival (OS) (p = 0.0076) than those without PIWIL1 expression. PIWIL2 and 4 were downregulated in tumor tissue in comparison to the normal tissue (p < 0.001) and the patients with lower levels of PIWIL4 had shorter TTR (p = 0.048) and OS (p = 0.033). In the multivariate analysis, PIWIL1 expression emerged as an independent prognostic marker. Using 5-Aza-dC treatment and bisulfite sequencing, we observed that PIWIL1 expression could be regulated in part by methylation. Finally, an in silico study identified a stem-cell expression signature associated with PIWIL1 expression.

Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

PubMed Central

Tompkins, Joshua D.; Jung, Marc; Chen, Chang-yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2016-01-01

The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors. PMID:26981572

Tissue Factor-Factor VIIa Complex Triggers Protease Activated Receptor 2-Dependent Growth Factor Release and Migration in Ovarian Cancer

PubMed Central

Chanakira, Alice; Westmark, Pamela R.; Ong, Irene M.; Sheehan, John P.

2017-01-01

Objective Enhanced tissue factor (TF) expression in epithelial ovarian cancer (EOC) is associated with aggressive disease. Our objective was to evaluate the role of the TF-factor VIIa-protease-activated receptor-2 (PAR-2) pathway in human EOC. Methods TCGA RNAseq data from EOC databases were analyzed for PAR expression. Cell and microparticle (MP) associated TF protein expression (Western blot) and MP-associated coagulant activity were determined in human EOC (SKOV-3, OVCAR-3 and CaOV-3) and control cell lines. PAR-1 and PAR-2 protein expression were similarly examined. The PAR dependence of VEGF-A release (ELISA) and chemotactic migration in response to FVIIa and cellular proliferation in response to thrombin was evaluated with small molecule antagonists. Results Relative mRNA expression consistently demonstrated PAR-2>PAR-1≫PAR-3/4 in multiple EOC datasets. Human EOC cell line lysates confirmed expression of TF, PAR-1 and PAR-2 proteins. MPs isolated from EOC cell lines demonstrated markedly enhanced (4–10 fold) TF coagulant activity relative to control cell lines. FVIIa induced a dose-dependent increase in VEGF-A release (2.5-3 fold) from EOC cell lines that was abrogated by the PAR-2 antagonist ENMD-1068. FVIIa treatment of CaOV-3 and OVCAR-3 cells resulted in increased chemotactic migration that was abolished by ENMD-1068. Thrombin induced dose-dependent EOC cell line proliferation was completely reversed by the PAR-1 antagonist vorapaxar. Small molecule antagonists had no effect on these phenotypes without protease present. Conclusions Enhanced activity of the TF-FVIIa-PAR-2 axis may contribute to the EOC progression via PAR-2 dependent signaling that supports an angiogenic and invasive phenotype and local thrombin generation supporting PAR-1 dependent proliferation. PMID:28148395

Genetic predictors of MEK dependence in non-small cell lung cancer.

PubMed

Pratilas, Christine A; Hanrahan, Aphrothiti J; Halilovic, Ensar; Persaud, Yogindra; Soh, Junichi; Chitale, Dhananjay; Shigematsu, Hisayuki; Yamamoto, Hiromasa; Sawai, Ayana; Janakiraman, Manickam; Taylor, Barry S; Pao, William; Toyooka, Shinichi; Ladanyi, Marc; Gazdar, Adi; Rosen, Neal; Solit, David B

2008-11-15

Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non-small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a "driver" mutation in the cells in which it is expressed, MEK inhibition in (V600E)BRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR.

Lysophosphatidic acid-induced ADAM12 expression mediates human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth.

PubMed

Do, Eun Kyoung; Kim, Young Mi; Heo, Soon Chul; Kwon, Yang Woo; Shin, Sang Hun; Suh, Dong-Soo; Kim, Ki-Hyung; Yoon, Man-Soo; Kim, Jae Ho

2012-11-01

Lysophosphatidic acid (LPA) is involved in mesenchymal stem cell-stimulated tumor growth in vivo. However, the molecular mechanism by which mesenchymal stem cells promote tumorigenesis remains elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induced the expression of ADAM12, a disintegrin and metalloproteases family member, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated ADAM12 expression was abrogated by pretreatment of hASCs with the LPA receptor 1 inhibitor Ki16425 or by small interfering RNA-mediated silencing of LPA receptor 1, suggesting a key role for the LPA-LPA receptor 1 signaling axis in A549 CM-stimulated ADAM12 expression. Silencing of ADAM12 expression using small interfering RNA or short hairpin RNA abrogated LPA-induced expression of both α-smooth muscle actin, a marker of carcinoma-associated fibroblasts, and ADAM12 in hASCs. Using a xenograft transplantation model of A549 cells, we demonstrated that silencing of ADAM12 inhibited the hASC-stimulated in vivo growth of A549 xenograft tumors and the differentiation of transplanted hASCs to α-smooth muscle actin-positive carcinoma-associated fibroblasts. LPA-conditioned medium from hASCs induced the adhesion of A549 cells and silencing of ADAM12 inhibited LPA-induced expression of extracellular matrix proteins, periostin and βig-h3, in hASCs and LPA-conditioned medium-stimulated adhesion of A549 cells. These results suggest a pivotal role for LPA-stimulated ADAM12 expression in tumor growth and the differentiation of hASCs to carcinoma-associated fibroblasts expressing α-smooth muscle actin, periostin, and βig-h3. Copyright © 2012 Elsevier Ltd. All rights reserved.

S-aryl-L-cysteine sulphoxides and related organosulphur compounds alter oral biofilm development and AI-2-based cell-cell communication.

PubMed

Kasper, S H; Samarian, D; Jadhav, A P; Rickard, A H; Musah, R A; Cady, N C

2014-11-01

To design and synthesize a library of structurally related, small molecules related to homologues of compounds produced by the plant Petiveria alliacea and determine their ability to interfere with AI-2 cell-cell communication and biofilm formation by oral bacteria. Many human diseases are associated with persistent bacterial biofilms. Oral biofilms (dental plaque) are problematic as they are often associated with tooth decay, periodontal disease and systemic disorders such as heart disease and diabetes. Using a microplate-based approach, a bio-inspired small molecule library was screened for anti-biofilm activity against the oral species Streptococcus mutans UA159, Streptococcus sanguis 10556 and Actinomyces oris MG1. To complement the static screen, a flow-based BioFlux microfluidic system screen was also performed under conditions representative of the human oral cavity. Several compounds were found to display biofilm inhibitory activity in all three of the oral bacteria tested. These compounds were also shown to inhibit bioluminescence by Vibrio harveyi and were thus inferred to be quorum sensing (QS) inhibitors. Due to the structural similarity of these compounds to each other, and to key molecules in AI-2 biosynthetic pathways, we propose that these molecules potentially reduce biofilm formation via antagonism of QS or QS-related pathways. This study highlights the potential for a non-antimicrobial-based strategy, focused on AI-2 cell-cell signalling, to control the development of dental plaque. Considering that many bacterial species use AI-2 cell-cell signalling, as well as the increased concern of the use of antimicrobials in healthcare products, such an anti-biofilm approach could also be used to control biofilms in environments beyond the human oral cavity. © 2014 The Society for Applied Microbiology.

Vascular tissue engineering by computer-aided laser micromachining.

PubMed

Doraiswamy, Anand; Narayan, Roger J

2010-04-28

Many conventional technologies for fabricating tissue engineering scaffolds are not suitable for fabricating scaffolds with patient-specific attributes. For example, many conventional technologies for fabricating tissue engineering scaffolds do not provide control over overall scaffold geometry or over cell position within the scaffold. In this study, the use of computer-aided laser micromachining to create scaffolds for vascular tissue networks was investigated. Computer-aided laser micromachining was used to construct patterned surfaces in agarose or in silicon, which were used for differential adherence and growth of cells into vascular tissue networks. Concentric three-ring structures were fabricated on agarose hydrogel substrates, in which the inner ring contained human aortic endothelial cells, the middle ring contained HA587 human elastin and the outer ring contained human aortic vascular smooth muscle cells. Basement membrane matrix containing vascular endothelial growth factor and heparin was to promote proliferation of human aortic endothelial cells within the vascular tissue networks. Computer-aided laser micromachining provides a unique approach to fabricate small-diameter blood vessels for bypass surgery as well as other artificial tissues with complex geometries.

Anticancer activity of Astragalus polysaccharide in human non-small cell lung cancer cells.

PubMed

Wu, Chao-Yan; Ke, Yuan; Zeng, Yi-Fei; Zhang, Ying-Wen; Yu, Hai-Jun

2017-01-01

We have reported that Chinese herbs Astragalus polysaccharide (APS) can inhibit nuclear factor kappaB (NF-κB) activity during the development of diabetic nephropathy in mice. NF-κB plays important roles in genesis, growth, development and metastasis of cancer. NF-κB is also involved in the development of treatment resistance in tumors. Here we investigated the antitumor activity of APS in human non-small cell lung cells (A549 and NCI-H358) and the related mechanisms of action. The dose-effect and time-effect of antitumor of APS were determined in human lung cancer cell line A549 and NCI-H358. The inhibition effect of APS on the P65 mRNA and protein was detected by reverse transcriptase-PCR (RT-PCR) and Western blot in A549 cells respectively. The inhibition effect of APS on the p50, CyclinD1 and Bcl-xL protein was detected by Western blot in A549 cells respectively. The effect of APS on NF-κB transcription activity was measured with NF-κB luciferase detection. Finally, the nude mice A549 xenograft was introduced to confirm the antitumor activity of APS in vivo. Cell viability detection results indicated that APS can inhibit the proliferation of human lung cancer cell line A549 and NCI-H358 in the concentration of 20 and 40 mg/mL. NF-κB activator Phorbol 12-myristate13-acetate (PMA) can attenuate the antitumor activity of APS in both cell lines, but NF-κB inhibitor BAY 11-7082 (Bay) can enhance the effect of APS in both cell lines. In vivo APS can delay the growth of A549 xenograft in BALB/C nude mice. APS can down-regulate the expression of P65 mRNA and protein of A549 cells and decrease the expression of p50, CyclinD1 and Bcl-xL protein. The luciferase detection showed that the APS could reduce the P65 transcription activity in A549 cells. PMA can partially alleviate the inhibition activity of P65 transcription activity of APS in A549 cells, and Bay can enhance the down-regulation of the P65 transcription activity induced by APS in A549 cells. APS has a significant antitumor activity in human lung cancer cells A549 and NCI-H358. NF-κB inhibition may mediate the antitumor effect.

Personalized drug discovery: HCA approach optimized for rare diseases at Tel Aviv University.

PubMed

Solmesky, Leonardo J; Weil, Miguel

2014-03-01

The Cell screening facility for personalized medicine (CSFPM) at Tel Aviv University in Israel is devoted to screening small molecules libraries for finding new drugs for rare diseases using human cell based models. The main strategy of the facility is based on smartly reducing the size of the compounds collection in similarity clusters and at the same time keeping high diversity of pharmacophores. This strategy allows parallel screening of several patient derived - cells in a personalized screening approach. The tested compounds are repositioned drugs derived from collections of phase III and FDA approved small molecules. In addition, the facility carries screenings using other chemical libraries and toxicological characterizations of nanomaterials.

Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells

PubMed Central

Chen, Yu-Ching; Chang, Hsun-Ming; Cheng, Jung-Chien; Tsai, Horng-Der; Wu, Cheng-Hsuan; Leung, Peter C.K.

2015-01-01

STUDY QUESTION Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell–cell communication in human granulosa cells? SUMMARY ANSWER TGF-β1 up-regulates Cx43 and increases gap junction intercellular communication activities (GJIC) in human granulosa cells, and this effect occurs via the activin receptor-like kinase (ALK)5-mediated Sma- and Mad-related protein (SMAD)2/3-SMAD4-dependent pathway. WHAT IS KNOWN ALREADY TGF-β1 and its receptors are expressed in human granulosa cells, and follicular fluid contains TGF-β1 protein. In human granulosa cells, Cx43 gap junctions play an important role in the development of follicles and oocytes. STUDY DESIGN, SIZE, DURATION This is an experimental study which was performed over a 1-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing IVF in an academic research center were used as the study models. Cx43 mRNA and protein expression levels were examined after exposure of SVOG cells to recombinant human TGF-β1. An activin/TGF-β type I receptor inhibitor, SB431542, and small interfering RNAs targeting ALK4, ALK5, SMAD2, SMAD3 and SMAD4 were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance. MAIN RESULTS AND THE ROLE OF CHANCE TGF-β1 treatment increased phosphorylation of SMAD2/3 (P < 0.0001) and up-regulated Cx43 mRNA and protein levels (P < 0.001) in SVOG cells and these stimulatory effects were abolished by the TGF-β type I receptor inhibitor SB431542. In addition, the up-regulatory effect of TGF-β1 on Cx43 expression (mRNA and protein) was confirmed in primary cultures of human granulosa-lutein cells (P < 0.05). The small interfering RNA-mediated knockdown of ALK5, but not ALK4, abolished the TGF-β1-induced phosphorylation of SMAD2/3 and the up-regulation of Cx43. Furthermore, knockdown of SMAD2/3 or the common SMAD, SMAD4, abolished the stimulatory effects of TGF-β1 on Cx43 expression in SVOG cells. The TGF-β1-induced up-regulation of Cx43 contributed to the increase of GJIC between SVOG cells (P < 0.001). LIMITATIONS, REASONS FOR CAUTION The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo. WIDER IMPLICATIONS OF THE FINDINGS Our studies represent the first comprehensive research of molecular mechanisms of TGF-β1 in the regulation of Cx43 expression and GJIC in human granulosa cells and demonstrate that TGF-β1 may play a crucial role in the local modulation of cell–cell communication. Deepening our understanding of the molecular determinants will offer important insights into ovarian physiology and lead to the development of potential therapeutic methods for fertility regulation. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by an operating grant from the Canadian Institutes of Health Research to P.C.K.L. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER NA. PMID:26202915

Caspase vinyl sulfone small molecule inhibitors prevent axonal degeneration in human neurons and reverse cognitive impairment in Caspase-6-overexpressing mice.

PubMed

Pakavathkumar, Prateep; Noël, Anastasia; Lecrux, Clotilde; Tubeleviciute-Aydin, Agne; Hamel, Edith; Ahlfors, Jan-Eric; LeBlanc, Andrea C

2017-02-28

The activation of the aspartate-specific cysteinyl protease, Caspase-6, is proposed as an early pathogenic event of Alzheimer disease (AD) and Huntington's disease. Caspase-6 inhibitors could be useful against these neurodegenerative diseases but most Caspase-6 inhibitors have been exclusively studied in vitro or show acute liver toxicity in humans. Here, we assessed vinyl sulfone small molecule peptide caspase inhibitors for potential use in vivo. The IC 50 of NWL vinyl sulfone small molecule caspase inhibitors were determined on Caspase-1 to 10, and Caspase-6-transfected human colon carcinoma HCT116 cells. Inhibition of Caspase-6-mediated axonal degeneration was assessed in serum-deprived or amyloid precursor protein-transfected primary human CNS neurons. Cellular toxicity was measured by phase contrast microscopy, mitochondrial and lactate dehydrogenase colorimetric activity assays, or flow cytometry. Caspase inhibition was measured by fluorogenic activity assays, fluorescence microscopy, and western blot analyses. The effect of inhibitors on age-dependent cognitive deficits in Caspase-6 transgenic mice was assessed by the novel object recognition task. Liquid chromatography coupled to tandem mass spectrometry assessed the blood-brain barrier permeability of inhibitors in Caspase-6 mice. Vinyl sulfone NWL-117 caspase inhibitor has a higher selectivity against Caspase-6, -4, -8, -9, and -10 whereas NWL-154 has higher selectivity against Caspase-6, -8, and -10. The half-maximal inhibitory concentrations (IC 50 ) of NWL-117 and NWL-154 is 192 nM and 100 nM against Caspase-6 in vitro, and 4.82 μM and 3.63 μM in Caspase-6-transfected HCT116 cells, respectively. NWL inhibitors are not toxic to HCT116 cells or to human primary neurons. NWL-117 and NWL-154 inhibit serum deprivation-induced Caspase-6 activity and prevent amyloid precursor protein-mediated neurite degeneration in human primary CNS neurons. NWL-117 crosses the blood brain barrier and reverses age-dependent episodic memory deficits in Caspase-6 mice. NWL peptidic vinyl methyl sulfone inhibitors are potent, non-toxic, blood-brain barrier permeable, and irreversible caspase inhibitors with neuroprotective effects in HCT116 cells, in primary human CNS neurons, and in Caspase-6 mice. These results highlight the therapeutic potential of vinyl sulfone inhibitors as caspase inhibitors against neurodegenerative diseases and sanction additional work to improve their selectivity against different caspases.

Lymphatic exosomes promote dendritic cell migration along guidance cues

PubMed Central

Brown, Markus; Johnson, Louise A.; Leone, Dario A.; Majek, Peter; Senfter, Daniel; Bukosza, Nora; Asfour, Gabriele; Langer, Brigitte; Parapatics, Katja; Hong, Young-Kwon; Bennett, Keiryn L.; Sixt, Michael

2018-01-01

Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments. PMID:29650776

Neuroactive substances in the human vestibular end organs.

PubMed

Usami, S; Matsubara, A; Shinkawa, H; Matsunaga, T; Kanzaki, J

1995-01-01

In order to evaluate the involvement of neuroactive substances in the human vestibular periphery, the immunocytochemical distribution of substance P (SP), calcitonin gene-related peptide (CGRP), and choline acetyltransferase (ChAT) was examined. SP-like immunoreactivity (LI) was present around and beneath sensory hair cells, probably corresponding to their afferent nerve endings. SP-LI was found predominantly in subpopulations of the primary afferents distributed in the peripheral region of the end organs. ChAT-LI and CGRP-LI were found throughout as small puncta below the hair cell layer, probably corresponding to efferent endings. The present results indicate that these neuroactive substances, previously described in animals, are also distributed in the human vestibular periphery, and almost certainly contribute to human vestibular function.

Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo

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

Lee, Miyoung; Jeong, Sang Young; Ha, Jueun

2014-04-18

Highlights: • hUCB-MSCs maintained low immunogenicity even after immune challenge in vitro. • Humanized NSG mice were established using human UCB CD34+ cells. • Repeated intravenous hUCB-MSC injection into mice did not lead to immune responses and adverse events. • Allogeneic hUCB-MSCs maintained low immunogenicity in vitro and in vivo. - Abstract: Evaluation of the immunogenicity of human mesenchymal stem cells (MSCs) in an allogeneic setting during therapy has been hampered by lack of suitable models due to technical and ethical limitations. Here, we show that allogeneic human umbilical cord blood derived-MSCs (hUCB-MSCs) maintained low immunogenicity even after immune challengemore » in vitro. To confirm these properties in vivo, a humanized mouse model was established by injecting isolated hUCB-derived CD34+ cells intravenously into immunocompromised NOD/SCID IL2γnull (NSG) mice. After repeated intravenous injection of human peripheral blood mononuclear cells (hPBMCs) or MRC5 cells into these mice, immunological alterations including T cell proliferation and increased IFN-γ, TNF-α, and human IgG levels, were observed. In contrast, hUCB-MSC injection did not elicit these responses. While lymphocyte infiltration in the lung and small intestine and reduced survival rates were observed after hPBMC or MRC5 transplantation, no adverse events were observed following hUCB-MSC introduction. In conclusion, our data suggest that allogeneic hUCB-MSCs have low immunogenicity in vitro and in vivo, and are therefore “immunologically safe” for use in allogeneic clinical applications.« less

Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes.

PubMed

Desch, A Nicole; Gibbings, Sophie L; Goyal, Rajni; Kolde, Raivo; Bednarek, Joe; Bruno, Tullia; Slansky, Jill E; Jacobelli, Jordan; Mason, Robert; Ito, Yoko; Messier, Elise; Randolph, Gwendalyn J; Prabagar, Miglena; Atif, Shaikh M; Segura, Elodie; Xavier, Ramnik J; Bratton, Donna L; Janssen, William J; Henson, Peter M; Jakubzick, Claudia V

2016-03-15

The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics.

Downregulation of long non-coding RNA LET predicts poor prognosis and increases Notch signaling in non-small cell lung cancer

PubMed Central

Li, Shengwen; Zhao, Hui; Li, Jianqiang; Zhang, Aizheng; Wang, Haibin

2018-01-01

Long non-coding RNAs (lncRNAs) have been found to be dysregulated in a variety of tumors. The lncRNA-Low Expression in Tumor (LET) is a recently identified lncRNA, but its expression pattern and biological significance in human non-small cell lung cancer (NSCLC) are still largely unknown. In this study, we found that lncRNA-LET was significantly downregulated in human NSCLC lung tissues and cell lines. Decreased lncRNA-LET expression was strongly associated with advanced tumor stages and poorer overall survival of NSCLC patients. Functionally, overexpression of lncRNA-LET in NSCLC H292 cells significantly suppressed cell proliferation, migration and invasion, and promoted cell cycle arrest and apoptosis, while knockdown of lncRNA-LET in NSCLC H1975 cells showed an opposite effect, pointing to a tumor-suppressive role for lncRNA-LET in NSCLC. Mechanistically, we demonstrated that lncRNA-LET overexpression significantly reduced the expression of Notch1 intracellular Domain (NICD1) in H292 cells while knockdown of lncRNA-LET increased NICD1 expression in H1975 cells. Similarly, NSCLC lung tissues with high levels of lncRNA-LET had lower NICD1 expression. Thus, our results provide a strong rationale for lncRNA-LET to be used as a prognostic indicator and a potent therapeutic target for NSCLC patients, and highlight a novel lncRNA-LET/Notch axis in regulating NSCLC cell fate and tumor progression. PMID:29416684

Loss in lung volume and changes in the immune response demonstrate disease progression in African green monkeys infected by small-particle aerosol and intratracheal exposure to Nipah virus.

PubMed

Cong, Yu; Lentz, Margaret R; Lara, Abigail; Alexander, Isis; Bartos, Christopher; Bohannon, J Kyle; Hammoud, Dima; Huzella, Louis; Jahrling, Peter B; Janosko, Krisztina; Jett, Catherine; Kollins, Erin; Lackemeyer, Matthew; Mollura, Daniel; Ragland, Dan; Rojas, Oscar; Solomon, Jeffrey; Xu, Ziyue; Munster, Vincent; Holbrook, Michael R

2017-04-01

Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that emerged in the late 1990s in Malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in Bangladesh and India. NiV infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of NiV contaminated foods. In the work presented here, the development of disease was investigated in the African Green Monkey (AGM) model following intratracheal (IT) and, for the first time, small-particle aerosol administration of NiV. This study utilized computed tomography (CT) and magnetic resonance imaging (MRI) to temporally assess disease progression. The host immune response and changes in immune cell populations over the course of disease were also evaluated. This study found that IT and small-particle administration of NiV caused similar disease progression, but that IT inoculation induced significant congestion in the lungs while disease following small-particle aerosol inoculation was largely confined to the lower respiratory tract. Quantitative assessment of changes in lung volume found up to a 45% loss in IT inoculated animals. None of the subjects in this study developed overt neurological disease, a finding that was supported by MRI analysis. The development of neutralizing antibodies was not apparent over the 8-10 day course of disease, but changes in cytokine response in all animals and activated CD8+ T cell numbers suggest the onset of cell-mediated immunity. These studies demonstrate that IT and small-particle aerosol infection with NiV in the AGM model leads to a severe respiratory disease devoid of neurological indications. This work also suggests that extending the disease course or minimizing the impact of the respiratory component is critical to developing a model that has a neurological component and more accurately reflects the human condition.

Loss in lung volume and changes in the immune response demonstrate disease progression in African green monkeys infected by small-particle aerosol and intratracheal exposure to Nipah virus

PubMed Central

Cong, Yu; Lentz, Margaret R.; Lara, Abigail; Alexander, Isis; Bartos, Christopher; Bohannon, J. Kyle; Hammoud, Dima; Huzella, Louis; Jahrling, Peter B.; Janosko, Krisztina; Jett, Catherine; Kollins, Erin; Lackemeyer, Matthew; Mollura, Daniel; Ragland, Dan; Rojas, Oscar; Solomon, Jeffrey; Xu, Ziyue; Munster, Vincent

2017-01-01

Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that emerged in the late 1990s in Malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in Bangladesh and India. NiV infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of NiV contaminated foods. In the work presented here, the development of disease was investigated in the African Green Monkey (AGM) model following intratracheal (IT) and, for the first time, small-particle aerosol administration of NiV. This study utilized computed tomography (CT) and magnetic resonance imaging (MRI) to temporally assess disease progression. The host immune response and changes in immune cell populations over the course of disease were also evaluated. This study found that IT and small-particle administration of NiV caused similar disease progression, but that IT inoculation induced significant congestion in the lungs while disease following small-particle aerosol inoculation was largely confined to the lower respiratory tract. Quantitative assessment of changes in lung volume found up to a 45% loss in IT inoculated animals. None of the subjects in this study developed overt neurological disease, a finding that was supported by MRI analysis. The development of neutralizing antibodies was not apparent over the 8–10 day course of disease, but changes in cytokine response in all animals and activated CD8+ T cell numbers suggest the onset of cell-mediated immunity. These studies demonstrate that IT and small-particle aerosol infection with NiV in the AGM model leads to a severe respiratory disease devoid of neurological indications. This work also suggests that extending the disease course or minimizing the impact of the respiratory component is critical to developing a model that has a neurological component and more accurately reflects the human condition. PMID:28388650

Exosomal particles secreted by prostate cancer cells are potent mRNA and protein vehicles for the interference of tumor and tumor environment.

PubMed

Rauschenberger, Lisa; Staar, Doreen; Thom, Kathleen; Scharf, Christian; Venz, Simone; Homuth, Georg; Schlüter, Rabea; Brandenburg, Lars-Ove; Ziegler, Patrick; Zimmermann, Uwe; Weitschies, Werner; Völker, Uwe; Lendeckel, Uwe; Walther, Reinhard; Burchardt, Martin; Stope, Matthias B

2016-03-01

Remodeling of the tumor environment and the modulation of tumor associated non-malignant cells are essential events in tumor progression. Exosomes are small membranous vesicles of 50-150 nm in diameter, which are secreted into the extracellular space and supposedly serve as vehicles for signal and effector molecules to modulate adjacent target cells. We characterized the mRNA and protein composition as well as cellular functions of prostate cancer cell-derived exosomes. Exosomes were prepared from prostate cancer cell culture supernatant by ultracentrifugation and subsequently characterized by dynamic light scattering and electron microscopy. Exosomal mRNA and protein composition were analyzed by DNA microarrays and gel electrophoresis coupled with mass spectrometry. Physiological effects of exosomes were studied by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release cell assays. Using a SILAC approach, putative uptake of exosomal human proteins in canine cells and canine de novo synthesis of proteins specified by exosome-transferred human mRNA was analyzed in MDCK cells via mass spectrometry. Preparations of exosomes revealed typical cup shaped particles of 150 nm in diameter. Analysis of mRNA and protein composition of exosomes exhibited a wide range of mRNA and protein species. Interestingly, the packaging of at least small proteins into exosomes was apparently unspecific, as shown with the example of two model proteins. In cell culture incubation experiments exosomal preparations of prostate cancer cells caused anti-proliferative effects. MS analysis revealed the uptake of exosomal human proteins into canine cells after 6 hr of incubation. The results reveal a distinct exosomal functionality in the modulation of the prostatic tumor adjacent environment. The multitude of translocated factors implies the induction of numerous effects in tumor-associated target cells, including impact on cellular growth. © 2015 Wiley Periodicals, Inc.

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

PubMed Central

Sanderson, I R; Ezzell, R M; Kedinger, M; Erlanger, M; Xu, Z X; Pringault, E; Leon-Robine, S; Louvard, D; Walker, W A

1996-01-01

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8755542

Aggregation of luteinizing hormone receptors in granulosa cells: a possible mechanism of desensitization to the hormone.

PubMed Central

Amsterdam, A; Berkowitz, A; Nimrod, A; Kohen, F

1980-01-01

The temporal relationship between redistribution of receptors to lutropin (luteinizing hormone)/human chorionic gonadotropin in cultured rat ovarian granulosa cells and the cellular response to hormonal challenge were studied. Visualization of receptor-bound human chorionic gonadotropin by indirect immunofluorescence, with hormone-specific antibodies after fixation with 2% formaldehyde, revealed the existence of small clusters around the entire cell circumference 5--20 min after exposure to the hormone at 37 degrees C. Such small receptor aggregates were also evident if hormone incubation was at 4 degrees C or if cells were fixed with 2% formaldehyde before incubation. Larger clusters were evident after prolonged incubation with the hormone (2--4 hr) at 37 degrees C. The later change coincided with diminished cyclic AMP accumulation in respose to challenge with fresh hormone. When the fixation step was omitted and antibodies to human chorionic gonadotropin were applied after hormonal binding, acceleration of both receptor clustering and the desensitization process was observed. This maneuver also induced capping of the hormone receptors. In contrast, monovalent Fab' fragments of the antibodies were without effect. Internalization of the bound hormone in lysosomes, and subsequent degradation, was evident 8 hr after hormonal application and was not accelerated by the antibodies. It is suggested that clustering of the luteinizing hormone receptors may play a role in cellular responsiveness to the hormone. Massive aggregation of the receptors may desensitize the cell by interferring with coupling to adenylate cyclase. Images PMID:6251459

Monitoring Ecological Impacts of Environmental Surface ...

EPA Pesticide Factsheets

Optimized cell-based metabolomics has been used to study the impacts of contaminants in surface waters on human and fish metabolomes. This method has proven to be resource- and time-effective, as well as sustainable for long term and large scale studies. In the current study, cell-based metabolomics is used to investigate the impacts of contaminants in surface waters on biological pathways in human and ecologically relevant cell lines. Water samples were collected from stream sites nationwide, where significant impacts have been estimated from the most potentially contaminated sources (i.e. waste water treatment plants, concentrated animal feeding operations, mining operations, and plant-based agricultural operations that use intensive chemical applications). Zebrafish liver cells (ZFL) were used to study exposure impacts on in vitro metabolomes. In addition, a small number of water samples were studied using two human cell lines (liver cells, HepG2 and brain cells, LN229). The cellular metabolites were profiled by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS). Detailed methods and results will be reported. Presented at SETAC North America 37th Annual Meeting

PlGF gene knockdown in human retinal pigment epithelial cells.

PubMed

Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Ahmadieh, Hamid; Rezaeikanavi, Mozhgan; Samiei, Shahram; Khalooghi, Keynoush

2011-04-01

To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells. Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs). At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs. Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells.

PubMed

Gong, Chenyuan; Ni, Zhongya; Yao, Chao; Zhu, Xiaowen; Ni, Lulu; Wang, Lixin; Zhu, Shiguo

2015-01-01

Recently, immunotherapy has shown a lot of promise in cancer treatment and different immune cell types are involved in this endeavor. Among different immune cell populations, NK cells are also an important component in unleashing the therapeutic activity of immune cells. Therefore, in order to enhance the tumoricidal activity of NK cells, identification of new small-molecule natural products is important. Despite the availability of different screening methods for identification of natural products, a simple, economic and high-throughput method is lacking. Hence, in this study, we have developed a high-throughput assay for screening and indentifying natural products that can enhance NK cell-mediated killing of cancer cells. We expanded human NK cell population from human peripheral blood mononuclear cells (PBMCs) by culturing these PBMCs with membrane-bound IL-21 and CD137L engineered K562 cells. Next, expanded NK cells were co-cultured with non-small cell lung cancer (NSCLC) cells with or without natural products and after 24 h of co-culturing, harvested supernatants were analyzed for IFN-γ secretions by ELISA method. We screened 502 natural products and identified that 28 candidates has the potential to induce IFN-γ secretion by NK cells to varying degrees. Among the 28 natural product candidates, we further confirmed and analyzed the potential of one molecule, andrographolide. It actually increased IFN-γ secretion by NK cells and enhanced NK cell-mediated killing of NSCLC cells. Our results demonstrated that this IFN-γ based high-throughput assay for screening of natural products for NK cell tumoricidal activity is a simple, economic and reliable method.

Flow cytometric monitoring of hormone receptor expression in human solid tumors

NASA Astrophysics Data System (ADS)

Krishan, Awtar

2002-05-01

Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.

Small Molecule Ice Recrystallization Inhibitors Enable Freezing of Human Red Blood Cells with Reduced Glycerol Concentrations

PubMed Central

Capicciotti, Chantelle J.; Kurach, Jayme D. R.; Turner, Tracey R.; Mancini, Ross S.; Acker, Jason P.; Ben, Robert N.

2015-01-01

In North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at −80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small molecule ice recrystallization inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystallization, a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addition of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol solution increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small molecule IRIs for the preservation of cells. PMID:25851700

Small molecule ice recrystallization inhibitors enable freezing of human red blood cells with reduced glycerol concentrations.

PubMed

Capicciotti, Chantelle J; Kurach, Jayme D R; Turner, Tracey R; Mancini, Ross S; Acker, Jason P; Ben, Robert N

2015-04-08

In North America, red blood cells (RBCs) are cryopreserved in a clinical setting using high glycerol concentrations (40% w/v) with slow cooling rates (~1°C/min) prior to storage at -80°C, while European protocols use reduced glycerol concentrations with rapid freezing rates. After thawing and prior to transfusion, glycerol must be removed to avoid intravascular hemolysis. This is a time consuming process requiring specialized equipment. Small molecule ice recrystallization inhibitors (IRIs) such as β-PMP-Glc and β-pBrPh-Glc have the ability to prevent ice recrystallization, a process that contributes to cellular injury and decreased cell viability after cryopreservation. Herein, we report that addition of 110 mM β-PMP-Glc or 30 mM β-pBrPh-Glc to a 15% glycerol solution increases post-thaw RBC integrity by 30-50% using slow cooling rates and emphasize the potential of small molecule IRIs for the preservation of cells.

Effect of Temporal Pattern of Radiation in Intensity Modulated Radiotherapy on Cell Cycle Progression and Apoptosis of ACHN Renal Cell Carcinoma Cell Line.

PubMed

Khorramizadeh, Maryam; Saberi, Alihossein; Tahmasebi-Birgani, Mohammadjavad; Shokrani, Parvaneh; Amouhedari, Alireza

The existence of a hypersensitive radiation response to doses below 1 Gy is well established for many normal and tumor cell lines. The aim of this study was to ascertain the impact of temporal pattern modeling IMRT on survival, cell cycle and apoptosis of human RCC cell line ACHN, so as to provide radiobiological basis for optimizing IMRT plans for this disease. The ACHN renal cell carcinoma cell line was used in this study. Impact of the triangle, V, small-large or large-small temporal patterns in the presence and absence of threshold dose of hyper-radiosensitivity at the beginning of patterns were studied using soft agarclonogenic assays. Cell cycle and apoptosis analysis were performed after irradiation with the temporal patterns. For triangle and small-large dose sequences, survival fraction was significantly reduced after irradiation with or without threshold dose of hyper-radiosensitivity at the beginning of the patterns. In all of the dose patterns, cell cycle distributions and the percentage of apoptotic cells at 24 h after irradiation with or without priming dose of hyper-radiosensitivity showed no significant difference. However, apoptotic cells were increased when beams with the smallest dose applied at the beginning of dose pattern like triangle and small-large dose sequence. These data show that the biologic effects of single fraction may differ in clinical settings depending on the size and sequence of the partial fractions. Doses at the beginning but not at the end of sequences may change cytotoxicity effects of radiation.

A novel small molecule, Rosline, inhibits growth and induces caspase-dependent apoptosis in human lung cancer cells A549 through a reactive oxygen species-dependent mechanism.

PubMed

Zhao, Ting; Feng, Yang; Jin, Wenling; Pan, Hui; Li, Haizhou; Zhao, Yang

2016-06-01

Chemical screening using synthetic small molecule libraries has provided a huge amount of novel active molecules. It generates lead compound for drug development and brings focus on molecules for mechanistic investigations on many otherwise intangible biological processes. In this study, using non-small cell lung cancer cell A549 to screen against a structurally novel and diverse synthetic small molecule library of 2,400 compounds, we identified a molecule named rosline that has strong anti-proliferation activity on A549 cells with a 50% cell growth inhibitory concentration (IC50 ) of 2.87 ± 0.39 µM. We showed that rosline treatment increased the number of Annexin V-positive staining cell, as well as G2/M arrest in their cell cycle progression. Further, we have demonstrated that rosline induces a decrease of mitochondrial membrane potential (Δφm ) and an increase of caspases 3/7 and 9 activities in A549 cells, although having no effect on the activity of caspase 8. Moreover, we found that rosline could induce the production of reactive oxygen species (ROS) and inhibit the phosphorylation of signaling molecule Akt in A549 cells. Alternatively, an antioxidant N-acetyl-L-cysteine (NAC) significantly attenuated rosline's effects on the mitochondrial membrane potential, caspases 3/7 and 9 activities, cell viabilities and the phosphorylation of Akt. Our results demonstrated that ROS played an important role in the apoptosis of A549 cells induced by rosline. © 2016 International Federation for Cell Biology.

Detection of DNA Damage by Space Radiation in Human Fibroblast Cells Flown on the International Space Station

NASA Technical Reports Server (NTRS)

Wu, Honglu; Lu, Tao; Wong, Michael; Beno, Jonathan; Countryman, Stefanie; Stodieck, Louis; Karouia, Fathi; Zhang, Ye

2015-01-01

Although charged particles in space have been detected with radiation detectors on board spacecraft since the early discovery of the Van Allen Belt, reports on effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation has been difficult due to the low dose and low dose rate nature of the radiation environment, and the difficulty in separating the radiation effects from microgravity and other space environmental factors. In astronauts, only a small number of changes, such as increased chromosome aberrations in lymphocytes and early onset of cataracts, attributed primarily to the exposure to space radiation. In a recent experiment, human fibroblast cells were flown on the International Space Station (ISS). Cells fixed on Days 3 and 14 after reaching orbit were analyzed for phosphorylation of a histone protein H2AX by immunofluorescent staining of cells, which is a widely used marker for DNA double strand breaks. The 3-dimensional gamma-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed a small fraction of foci that were larger and displayed a track pattern in the flight samples in comparison to the ground control. Human fibroblast cells were also exposed to low dose rate gamma rays, as well as to protons and Fe ions. Comparison of the pattern and distribution of the foci after gamma ray and charged particle exposure to our flight results confirmed that the foci found in the flown cells were indeed induced by space radiation.

Human umbilical cord blood-derived mesenchymal stromal cells and small intestinal submucosa hydrogel composite promotes combined radiation-wound healing of mice.

PubMed

Lee, Changsun; Shim, Sehwan; Jang, Hyosun; Myung, Hyunwook; Lee, Janet; Bae, Chang-Hwan; Myung, Jae Kyung; Kim, Min-Jung; Lee, Seung Bum; Jang, Won-Suk; Lee, Sun-Joo; Kim, Hwi-Yool; Lee, Seung-Sook; Park, Sunhoo

2017-09-01

Mesenchymal stromal cells (MSCs) are a promising agent for treating impaired wound healing, and their therapeutic potential may be enhanced by employing extracellular matrix scaffolds as cell culture scaffolds or transplant cell carriers. Here, we evaluated the effect of human umbilical cord blood-derived (hUCB)-MSCs and a porcine small intestinal submucosa (SIS)-derived extracellular matrix scaffold in a combined radiation-wound mouse model of impaired wound healing. hUCB-MSCs and SIS hydrogel composite was applied to the excisional wound of whole-body irradiated mice. Assessment of wound closing and histological evaluation were performed in vivo. We also cultured hUCB-MSCs on SIS gel and examined the angiogenic effect of conditioned medium on irradiated human umbilical vein endothelial cells (HUVECs) in vitro. hUCB-MSCs and SIS hydrogel composite treatment enhanced wound healing and angiogenesis in the wound site of mice. Conditioned medium from hUCB-MSCs cultured on SIS hydrogel promoted the chemotaxis of irradiated HUVECs more than their proliferation. The secretion of angiogenic growth factors hepatocyte growth factor, vascular endothelial growth factor-A and angiopoietin-1 from hUCB-MSCs was significantly increased by SIS hydrogel, with HGF being the predominant angiogenic factor of irradiated HUVECs. Our results suggest that the wound healing effect of hUCB-MSCs is enhanced by SIS hydrogel via a paracrine factor-mediated recruitment of vascular endothelial cells in a combined radiation-wound mouse model. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Prostasomes: Their Characterisation: Implications for Human Reproduction: Prostasomes and Human Reproduction.

PubMed

Ronquist, Gunnar

2015-01-01

The prostate is a principal accessory genital gland that is vital for normal fertility. Epithelial cells lining the prostate acini release in a defined fashion (exocytosis) organellar nanosized structures named prostasomes. They are involved in the protection of sperm cells against immune response in the female reproductive tract by modulating the complement system and by inhibiting monocyte and neutrophil phagocytosis and lymphocyte proliferation. The immunomodulatory function most probably involves small non-coding RNAs present in prostasomes. Prostasomes have also been proposed to regulate the timing of sperm cell capacitation and induction of the acrosome reaction, since they are rich in various transferable bioactive molecules (e.g. receptors and enzymes) that promote the fertilising ability of sperm cells. Antigenicity of sperm cells has been well documented and implicated in involuntary immunological infertility of human couples, and antisperm antibodies (ASA) occur in several body fluids. The propensity of sperm cells to carry attached prostasomes suggests that they are a new category of sperm antigens. Circulating human ASA recognise prostasomes, and among 12 identified prostasomal antigens, prolactin- inducible protein (95 %) and clusterin (85 %) were immunodominant at the expense of the other 10 that were sporadically occurring.

Accelerated high-yield generation of limb-innervating motor neurons from human stem cells

PubMed Central

Amoroso, Mackenzie W.; Croft, Gist F.; Williams, Damian J.; O’Keeffe, Sean; Carrasco, Monica A.; Davis, Anne R.; Roybon, Laurent; Oakley, Derek H.; Maniatis, Tom; Henderson, Christopher E.; Wichterle, Hynek

2013-01-01

Human pluripotent stem cells are a promising source of differentiated cells for developmental studies, cell transplantation, disease modeling, and drug testing. However, their widespread use even for intensely studied cell types like spinal motor neurons is hindered by the long duration and low yields of existing protocols for in vitro differentiation and by the molecular heterogeneity of the populations generated. We report a combination of small molecules that within 3 weeks induce motor neurons at up to 50% abundance and with defined subtype identities of relevance to neurodegenerative disease. Despite their accelerated differentiation, motor neurons expressed combinations of HB9, ISL1 and column-specific markers that mirror those observed in vivo in human fetal spinal cord. They also exhibited spontaneous and induced activity, and projected axons towards muscles when grafted into developing chick spinal cord. Strikingly, this novel protocol preferentially generates motor neurons expressing markers of limb-innervating lateral motor column motor neurons (FOXP1+/LHX3−). Access to high-yield cultures of human limb-innervating motor neuron subtypes will facilitate in-depth study of motor neuron subtype-specific properties, disease modeling, and development of large-scale cell-based screening assays. PMID:23303937

Transforming growth factor β-activated kinase 1 inhibitor suppresses the proliferation in triple-negative breast cancer through TGF-β/TGFR pathway.

PubMed

Zhang, Liangyu; Fu, Zelong; Li, Xia; Tang, Haitao; Luo, Jiesi; Zhang, Dehui; Zhuang, Yongzhi; Han, Zhiyang; Yin, Mingzhu

2017-09-01

Breast cancer is one of the most invasive cancer types in female population. The functional activity of Transforming growth factor β-activated kinase 1 (TAK1) in breast cancer progression increasingly attracts attention as it provides a potential target for antibreast cancer drug development. However, the fundamental role of TAK1 for triple-negative breast cancer (TNBC) progression and the effect of potential anti-TAK1 drug candidate needs to be further evaluated. Herein, we focused on the role of TAK1 in human breast cancer cells, and we hypothesized that the inhibition of TAK1 activation can repress the growth of human TNBC cells. We found that the TAK1 is robustly activated within cancer cell population of clinic-derived TNBC samples and the human breast cancer cell lines in culture. Furthermore, we determined the effect of 5Z-7-oxozeaenol (5Z-O), a TAK1-specific small molecule inhibitor, on proliferation of human TNBC cell line. 5Z-O treatment significantly suppressed the proliferation of human TNBC cells. Collectively, these demonstrate the role of TAK1 in human breast cancer and the antiproliferate effect of TAK1 inhibitor. Our study sets the stage for further research on TAK1 as a promising target for development of anti-TNBC drugs and therapeutic strategies. © 2017 John Wiley & Sons A/S.

Embryonic stem cell research: one small step for science or one giant leap back for mankind?

PubMed

Erwin, Consuelo G

2003-01-01

At the forefront of modern debate over the ethical use of biotechnology is embryonic stem cell research. In this poignant analysis of its legitimacy, the author examines the history of this research in light of the United States' policy favoring the protection of human beings over scientific progress. Stem cells, which can divide in culture to create specialized cells in the human body, possess significant potential for curing disease, particularly when taken from human embryos. However, as evidenced by the research atrocities committed under the Nazi regime, the benefits of human research do not come without a cost to humanity. Recognizing this, the later trial of these scientists produced the Nuremberg Code, a set of natural law principles guiding future research on humans that continues to influence health policy decisions. Drawing on this background, the author first considers the appropriate legal status for a human embryo. Biologically, the characteristics of a human embryo place it between human tissue and a constitutional person. Judicially, the answer is even less clear. The author analyzes case law in the context of abortion and in vitro fertilization, as well as classifications by the common law, state legislation, and the National Bioethics Advisory Commission, to conclude that a human embryo should be subject to the same legal and ethical restrictions as any other "human subject." Accordingly, the author argues that embryonic stem cell research violates the ethical standards and purposes of the Nuremberg Code and should be banned by federal legislation. Such a prohibition will fulfill the societal policy choice of protecting potential life and vulnerable human subjects.

The Role of T-Cell Leukemia Translocation-Associated Gene Protein in Human Tumorigenesis and Osteoclastogenesis

PubMed Central

Kotake, Shigeru; Yago, Toru; Kawamoto, Manabu; Nanke, Yuki

2012-01-01

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-κB ligand (RANKL), TNF-α, IL-6, IL-17, and IFN-γ. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in regulating bone resorption. In 2009, we demonstrated that novel peptides from T-cell leukemia translocation-associated gene (TCTA) protein expressed in synovial tissues from patients with RA inhibit human osteoclastogenesis, preventing cellular fusion via the interaction between TCTA protein and a putative counterpart molecule. Only a few studies on the role of TCTA protein have been reported. Genomic Southern blots demonstrated a reduced TCTA signal in three of four small cell lung cancer cell lines, suggesting the loss of one of the two copies of the gene. In the current paper, we reviewed the roles of TCTA protein in lung cancer cell lines and human osteoclastogenesis. PMID:22174563

U-937 Toxicity Testing of Lunar Dust Stimulant (JSC-1A-vf)

NASA Technical Reports Server (NTRS)

Bales, Kristyn; Hammond, Dianne; Wallace, William; Jeevarajan, Antony

2007-01-01

With NASA planning to extend the human presence to the moon by 2020, the dangers of the lunar environment must be assessed and appropriate countermeasures must be developed. Possible toxic effects of the lunar dust are of particular importance to human health because of the dust's chemical composition, reactivity, and small size. This project focuses on the toxicity of lunar dust stimulant (JSC-1A-vf), in both its active and passive forms, using U-937 human monocyte cells. Simulant was mechanically activated from its passive form by grinding, and its ability to produce hydroxyl radicals was determined. To test for toxicity, active and passivated simulant was diluted in media and applied to the cells for various time periods. Toxicity was then estimated using flow cytometry on the Guava Personal Cell Analysis system. Preliminary results suggest that passivated stimulant is slightly toxic, with an increase in toxicity for activated stimulant. Toxicity results may be affected by cell lysing behavior and quenching of hydroxyl radical production by the cell media.

Characterization of AQX-1125, a small-molecule SHIP1 activator

PubMed Central

Stenton, Grant R; Mackenzie, Patrick Tam, Lloyd F; Cross, Jennifer L; Harwig, Curtis; Raymond, Jeffrey; Toews, Judy; Wu, Joyce; Ogden, Nancy; MacRury, Thomas; Szabo, Csaba

2013-01-01

Background The SH2-containing inositol-5′-phosphatase 1 (SHIP1) metabolizes PI(3,4,5)P3 to PI(3,4)P2. SHIP1-deficient mice exhibit progressive inflammation. Pharmacological activation of SHIP1 is emerging as a potential therapy for pulmonary inflammatory diseases. Here we characterize the efficacy of AQX-1125, a small-molecule SHIP1 activator currently in clinical development. Experimental Approach The effects of AQX-1125 were tested in several in vitro assays: on enzyme catalytic activity utilizing recombinant human SHIP1, on Akt phosphorylation in SHIP1-proficient and SHIP1-deficient cell lines, on cytokine release in murine splenocytes, on human leukocyte chemotaxis using modified Boyden chambers and on β-hexosaminidase release from murine mast cells. In addition, pharmacokinetic and drug distribution studies were performed in rats and dogs. Results AQX-1125 increased the catalytic activity of human recombinant SHIP1, an effect, which was absent after deletion of the C2 region. AQX-1125 inhibited Akt phosphorylation in SHIP1-proficient but not in SHIP1-deficient cells, reduced cytokine production in splenocytes, inhibited the activation of mast cells and inhibited human leukocyte chemotaxis. In vivo, AQX-1125 exhibited >80% oral bioavailability and >5 h terminal half-life. Conclusions Consistent with the role of SHIP1 in cell activation and chemotaxis, the SHIP1 activator AQX-1125 inhibits Akt phosphorylation, inflammatory mediator production and leukocyte chemotaxis in vitro. The in vitro effects and the pharmacokinetic properties of the compound make it a suitable candidate for in vivo testing in various models of inflammation. Linked Article This article is accompanied by Stenton et al., pp. 1519–1529 of this issue. To view this article visit http://dx.doi.org/10.1111/bph.12038 PMID:23121445

Systematic screening identifies dual PI3K and mTOR inhibition as a conserved therapeutic vulnerability in osteosarcoma

PubMed Central

Gupte, Ankita; Baker, Emma K.; Wan, Soo-San; Stewart, Elizabeth; Loh, Amos; Shelat, Anang A.; Gould, Cathryn M.; Chalk, Alistair M.; Taylor, Scott; Lackovic, Kurt; Karlström, Åsa; Mutsaers, Anthony J.; Desai, Jayesh; Madhamshettiwar, Piyush B.; Zannettino, Andrew CW.; Burns, Chris; Huang, David CS.; Dyer, Michael A.; Simpson, Kaylene J.; Walkley, Carl R.

2015-01-01

Purpose Osteosarcoma (OS) is the most common cancer of bone occurring mostly in teenagers. Despite rapid advances in our knowledge of the genetics and cell biology of OS, significant improvements in patient survival have not been observed. The identification of effective therapeutics has been largely empirically based. The identification of new therapies and therapeutic targets are urgently needed to enable improved outcomes for OS patients. Experimental Design We have used genetically engineered murine models of human OS in a systematic, genome wide screen to identify new candidate therapeutic targets. We performed a genome wide siRNA screen, with or without doxorubicin. In parallel a screen of therapeutically relevant small molecules was conducted on primary murine and primary human OS derived cell cultures. All results were validated across independent cell cultures and across human and mouse OS. Results The results from the genetic and chemical screens significantly overlapped, with a profound enrichment of pathways regulated by PI3K and mTOR pathways. Drugs that concurrently target both PI3K and mTOR were effective at inducing apoptosis in primary OS cell cultures in vitro in both human and mouse OS, while specific PI3K or mTOR inhibitors were not effective. The results were confirmed with siRNA and small molecule approaches. Rationale combinations of specific PI3K and mTOR inhibitors could recapitulate the effect on OS cell cultures. Conclusions The approaches described here have identified dual inhibition of the PI3K/mTOR pathway as a sensitive, druggable target in OS and provide rationale for translational studies with these agents. PMID:25862761

CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation.

PubMed

Sunday, M E; Hua, J; Torday, J S; Reyes, B; Shipp, M A

1992-12-01

The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth.

A mixture of amino acids and other small molecules present in the serum suppresses the growth of murine and human tumors in vivo

PubMed Central

Kulcsár, Gyula; Gaál, Dezső; Kulcsár, Péter I; Schulcz, Ákos; Czömpöly, Tamás

2013-01-01

Previously we have hypothesized that the small molecules which are selectively accumulated in cancer cells might participate in a non-immunological antitumor surveillance mechanism. We demonstrated earlier that a mixture of experimentally selected substances (“active mixture”, AM: l-arginine, l-histidine, l-methionine, l-phenylalanine, l-tyrosine, l-tryptophan, l-ascorbate, d-biotin, pyridoxine, riboflavin, adenine, l(-)malate) possesses a selective toxic effect in vitro on a variety of tumor cell lines, and we have shown that the AM selectively induces apoptosis of cancer cells in vitro. To explore the in vivo significance of our earlier findings we examined the antitumor effect of AM in Colon 26 murine colorectal adenocarcinoma, B16 murine melanoma, MXT murine mammary carcinoma, S180 murine sarcoma, P388 murine lymphoid leukemia, HL-60 human promyeloid leukemia, PC-3 human prostate carcinoma, and HT-29 human colon carcinoma tumor models. Treatment of tumor bearing mice with AM inhibited the growth of the tumors investigated, with an inhibitory effect ranging from 40 to 69%. The AM had a comparable antitumor effect with 5-fluorouracil and cisplatin in the Colon-26 tumor model, and combined treatment with AM and 5-fluorouracil or cisplatin resulted in an enhanced tumor growth inhibitory effect. The AM induced apoptosis through the mitochondrial pathway and induced G1 arrest in PC-3 cells and increased the number of apoptotic cells in PC-3 xenografts. These findings suggest that the AM might offer an interesting perspective in the treatment of cancer and in combination with other treatments may offer hope for a more effective cancer therapy. PMID:22858865

A mixture of amino acids and other small molecules present in the serum suppresses the growth of murine and human tumors in vivo.

PubMed

Kulcsár, Gyula; Gaál, Dezső; Kulcsár, Péter I; Schulcz, Ákos; Czömpöly, Tamás

2013-03-01

Previously we have hypothesized that the small molecules which are selectively accumulated in cancer cells might participate in a non-immunological antitumor surveillance mechanism. We demonstrated earlier that a mixture of experimentally selected substances ("active mixture", AM: L-arginine, L-histidine, L-methionine, L-phenylalanine, L-tyrosine, L-tryptophan, L-ascorbate, D-biotin, pyridoxine, riboflavin, adenine, L(-)malate) possesses a selective toxic effect in vitro on a variety of tumor cell lines, and we have shown that the AM selectively induces apoptosis of cancer cells in vitro. To explore the in vivo significance of our earlier findings we examined the antitumor effect of AM in Colon 26 murine colorectal adenocarcinoma, B16 murine melanoma, MXT murine mammary carcinoma, S180 murine sarcoma, P388 murine lymphoid leukemia, HL-60 human promyeloid leukemia, PC-3 human prostate carcinoma, and HT-29 human colon carcinoma tumor models. Treatment of tumor bearing mice with AM inhibited the growth of the tumors investigated, with an inhibitory effect ranging from 40 to 69%. The AM had a comparable antitumor effect with 5-fluorouracil and cisplatin in the Colon-26 tumor model, and combined treatment with AM and 5-fluorouracil or cisplatin resulted in an enhanced tumor growth inhibitory effect. The AM induced apoptosis through the mitochondrial pathway and induced G1 arrest in PC-3 cells and increased the number of apoptotic cells in PC-3 xenografts. These findings suggest that the AM might offer an interesting perspective in the treatment of cancer and in combination with other treatments may offer hope for a more effective cancer therapy. Copyright © 2012 UICC.

Interaction between human BAP31 and respiratory syncytial virus small hydrophobic (SH) protein

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

Li, Yan; Jain, Neeraj; Limpanawat, Suweeraya

2015-08-15

The small hydrophobic (SH) protein is a short channel-forming polypeptide encoded by the human respiratory syncytial virus (hRSV). Deletion of SH protein leads to the viral attenuation in mice and primates, and delayed apoptosis in infected cells. We have used a membrane-based yeast two-hybrid system (MbY2H) and a library from human lung cDNA to detect proteins that bind SH protein. This led to the identification of a membrane protein, B-cell associated protein 31 (BAP31). Transfected SH protein co-localizes with transfected BAP31 in cells, and pulls down endogenous BAP31. Titration of purified C-terminal endodomain of BAP31 against isotopically labeled SH proteinmore » in detergent micelles suggests direct interaction between the two proteins. Given the key role of BAP31 in protein trafficking and its critical involvement in pro- and anti-apoptotic pathways, this novel interaction may constitute a potential drug target. - Highlights: • A yeast two-hybrid system (MbY2H) detected BAP31 as a binder of RSV SH protein. • Transfected SH and BAP31 co-localize in lung epithelial cells. • Endogenous BAP31 is pulled down by RSV SH protein. • BAP31 endodomain interacts with the N-terminal α-helix of SH protein in micelles. • This interaction is proposed to be a potential drug target.« less

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

PubMed

Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

2006-05-01

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

Protection and mechanism of action of a novel human respiratory syncytial virus vaccine candidate based on the extracellular domain of small hydrophobic protein

PubMed Central

Schepens, Bert; Sedeyn, Koen; Vande Ginste, Liesbeth; De Baets, Sarah; Schotsaert, Michael; Roose, Kenny; Houspie, Lieselot; Van Ranst, Marc; Gilbert, Brian; van Rooijen, Nico; Fiers, Walter; Piedra, Pedro; Saelens, Xavier

2014-01-01

Infections with human respiratory syncytial virus (HRSV) occur globally in all age groups and can have devastating consequences in young infants. We demonstrate that a vaccine based on the extracellular domain (SHe) of the small hydrophobic (SH) protein of HRSV, reduced viral replication in challenged laboratory mice and in cotton rats. We show that this suppression of viral replication can be transferred by serum and depends on a functional IgG receptor compartment with a major contribution of FcγRI and FcγRIII. Using a conditional cell depletion method, we provide evidence that alveolar macrophages are involved in the protection by SHe-specific antibodies. HRSV-infected cells abundantly express SH on the cell surface and are likely the prime target of the humoral immune response elicited by SHe-based vaccination. Finally, natural infection of humans and experimental infection of mice or cotton rats does not induce a strong immune response against HRSV SHe. Using SHe as a vaccine antigen induces immune protection against HRSV by a mechanism that differs from the natural immune response and from other HRSV vaccination strategies explored to date. Hence, HRSV vaccine candidates that aim at inducing protective neutralizing antibodies or T-cell responses could be complemented with a SHe-based antigen to further improve immune protection. PMID:25298406

Acquired Epidermodysplasia Verruciformis Associated with Human Papilloma Virus Type 14 in a Small Bowel Transplanted Child--A Case Report.

PubMed

Hirschman, Derek; Tacastacas, Joselin; Rady, Peter L; Tyring, Stephen K; Cooper, Kevin; Honda, Kord

2016-01-01

A 3-year-old African American girl taking sirolimus and tacrolimus for a small bowel transplantation presented with hypopigmented macules and papules throughout her trunk. A biopsy diagnosed epidermodysplasia verruciformis (EV) that was found to be associated with human papillomavirus (HPV) type 14 according to polymerase chain reaction analysis. There are few cases of acquired EV in the setting of organ transplantation. Although there is no standardized treatment for acquired EV, prevention and surveillance for transformation to squamous cell carcinoma are primary concerns. © 2015 Wiley Periodicals, Inc.

MicroRNA-429 induces tumorigenesis of human non-small cell lung cancer cells and targets multiple tumor suppressor genes

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

Lang, Yaoguo; Xu, Shidong; Ma, Jianqun

2014-07-18

Highlights: • MiR-429 expression is upregulated in non-small cell lung cancer (NSCLC). • MiR-429 inhibits PTEN, RASSF8 and TIMP2 expression. • MiR-429 promotes metastasis and proliferation. • We report important regulatory mechanisms involved in NSCLC progression. • MiR-429 is a potential therapeutic target and diagnostic marker. - Abstract: Lung cancer is the major cause of cancer death globally. MicroRNAs are evolutionally conserved small noncoding RNAs that are critical for the regulation of gene expression. Aberrant expression of microRNA (miRNA) has been implicated in cancer initiation and progression. In this study, we demonstrated that the expression of miR-429 are often upregulatedmore » in non-small cell lung cancer (NSCLC) compared with normal lung tissues, and its expression level is also increased in NSCLC cell lines compared with normal lung cells. Overexpression of miR-429 in A549 NSCLC cells significantly promoted cell proliferation, migration and invasion, whereas inhibition of miR-429 inhibits these effects. Furthermore, we demonstrated that miR-429 down-regulates PTEN, RASSF8 and TIMP2 expression by directly targeting the 3′-untranslated region of these target genes. Taken together, our results suggest that miR-429 plays an important role in promoting the proliferation and metastasis of NSCLC cells and is a potential target for NSCLC therapy.« less

Combination of Near Infrared Light-Activated Photodynamic Therapy Mediated by Indocyanine Green with Etoposide to Treat Non-Small-Cell Lung Cancer

PubMed Central

Luo, Ting; Zhang, Qinrong; Lu, Qing-Bin

2017-01-01

Indocyanine green (ICG) has been reported as a potential near-infrared (NIR) photosensitizer for photodynamic therapy (PDT) of cancer. However the application of ICG-mediated PDT is both intrinsically and physiologically limited. Here we report a combination of ICG-PDT with a chemotherapy drug etoposide (VP-16), aiming to enhance the anticancer efficacy, to circumvent limitations of PDT using ICG, and to reduce side effects of VP-16. We found in controlled in vitro cell-based assays that this combination is effective in killing non-small-cell lung cancer cells (NSCLC, A549 cell line). We also found that the combination of ICG-PDT and VP-16 exhibits strong synergy in killing non-small-cell lung cancer cells partially through inducing more DNA double-strand breaks (DSBs), while it has a much weaker synergy in killing human normal cells (GM05757). Furthermore, by studying the treatment sequence dependence and the cytotoxicity of laser-irradiated mixtures of ICG and VP-16, we found that the observed synergy involves direct/indirect reactions between ICG and VP-16. We further propose that there exists an electron transfer reaction between ICG and VP-16 under irradiation. This study therefore shows the anticancer efficacy of ICG-PDT combined with VP-16. These findings suggest that ICG-mediated PDT may be applied in combination with the chemotherapy drug VP-16 to treat some cancers, especially the non-small-cell lung cancer. PMID:28587258

Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions

PubMed Central

Kuligina, Elena V.; Bariakin, Dmitry N.; Kozlov, Vadim V.; Richter, Vladimir A.; Semenov, Dmitry V.

2017-01-01

Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000g and 160,000g, and vesicle-depleted plasma supernatant of healthy donors and non-small cell lung cancer (NSCLC) patients. It was determined that 16,000g blood plasma vesicles were enriched with cell-free mitochondria and with a set of mitochondrial RNAs. The variable RNA set of blood plasma 160,000g pellets reflected the prominent contribution of U1, U5, and U6 small nuclear RNAs' fragments and at the same time was characterized by a remarkable depletion of small nucleolar RNAs. Besides microRNAs, the variety of fragments of mRNAs and snoRNAs dominated in the set of circulating RNAs differentially expressed in blood fractions of NSCLC patients. Taken together, our data emphasize that not only extracellular microRNAs but also circulating fragments of messenger and small nuclear/nucleolar RNAs represent prominent classes of circulating regulatory ncRNAs as well as promising circulating biomarkers for the development of disease diagnostic approaches. PMID:28127559

Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

PubMed

Tong, Zhixiang; Martyn, Keir; Yang, Andy; Yin, Xiaolei; Mead, Benjamin E; Joshi, Nitin; Sherman, Nicholas E; Langer, Robert S; Karp, Jeffrey M

2018-02-01

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

PubMed

Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

CPS1 maintains pyrimidine pools and DNA synthesis in KRAS/LKB1-mutant lung cancer cells. | Office of Cancer Genomics

Cancer.gov

Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumor suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small-cell lung cancer(NSCLC). Concurrent occurrence of oncogenic KRAS and loss of LKB1 (KL) in cells specifies aggressive oncological behavior. Here we show that human KL cells and tumors share metabolic signatures of perturbed nitrogen handling.

Factors influencing alternative splice site utilization in vivo.

PubMed Central

Fu, X Y; Manley, J L

1987-01-01

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA. Images PMID:3029566

Fisetin induces apoptosis and endoplasmic reticulum stress in human non-small cell lung cancer through inhibition of the MAPK signaling pathway.

PubMed

Kang, Kyoung Ah; Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Ryu, Yea Seong; Oh, Min Chang; Kwon, Taeg Kyu; Chae, Sungwook; Hyun, Jin Won

2016-07-01

Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid compound, is currently being investigated for its anticancer effect in various cancer models, including lung cancer. Recent studies show that fisetin induces cell growth inhibition and apoptosis in the human non-small cell lung cancer line NCI-H460. In this study, we investigated whether fisetin can induce endoplasmic reticulum (ER) stress-mediated apoptosis in NCI-H460 cells. Fisetin induced mitochondrial reactive oxygen species (ROS) and characteristic signs of ER stress: ER staining; mitochondrial Ca(2+) overload; expression of ER stress-related proteins; glucose-regulated protein (GRP)-78, phosphorylation of protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylation of eukaryotic initiation factor-2 α subunit; cleavage of activating transcription factor-6; phosphorylation of inositol-requiring kinase-1 and splicing of X-box transcription factor-1; induction of C/EBP homologous protein and cleaved caspase-12. siRNA-mediated knockdown of CHOP and ATF-6 attenuated fisetin-induced apoptotic cell death. In addition, fisetin induced phosphorylation of ERK, JNK, and p38 MAPK. Moreover, silencing of the MAPK signaling pathway prevented apoptotic cell death. In summary, our results indicate that, in NCI-H460 cells, fisetin induces apoptosis and ER stress that is mediated by induction of the MAPK signaling pathway.

Sulforaphane Induced Apoptosis via Promotion of Mitochondrial Fusion and ERK1/2-Mediated 26S Proteasome Degradation of Novel Pro-survival Bim and Upregulation of Bax in Human Non-Small Cell Lung Cancer Cells.

PubMed

Geng, Yang; Zhou, Yan; Wu, Sai; Hu, Yabin; Lin, Kai; Wang, Yalin; Zheng, Zhongnan; Wu, Wei

2017-01-01

Previous studies in our laboratory showed that sulforaphane (SFN) induced apoptosis by sustained activation of extracellular regulated protein kinases 1/2 (ERK1/2). However, the underlying mechanisms associated with SFN-induced apoptosis and downstream cascades which are modulated by ERK1/2 were not elucidated. Herein we demonstrated for the first time that alteration of mitochondrial dynamics contributed to SFN-induced apoptosis in human non-small cell lung cancer (NSCLC) cells. Reports showed that protein Bim not only induced apoptosis but also promoted proliferation under certain circumstances. We found that Bim was related to cell growth in NSCLC cells. Pro-survival Bim downregulation was shown to induce apoptosis in response to SFN. Further, Using the ERK1/2 inhibitor, PD98059, we found that SFN upregulated Bax and downregulated Bim through the ERK1/2-dependent signaling pathway. Furthermore, SFN activated ERK1/2 to increase 26S proteasome activity to degrade Bim, while the proteasome inhibitor MG132 reversed this effect. Therefore, SFN phosphorylated ERK1/2 and activated the proteasome system leading to the degradation of Bim, which contributed to apoptosis in NSCLC cells. These findings provided a novel insight into SFN-related therapeutics in cancer treatment.

Modulation of the response of prostate cancer cell lines to cisplatin treatment using small interfering RNA.

PubMed

Parra, Eduardo; Ferreira, Jorge

2013-10-01

Cisplatin is one of the most effective and widely used chemotherapeutic agents against several types of human cancers. However, the underlying mechanisms of action are not fully understood. We aimed to investigate the possible molecular mechanism(s) of acquired chemoresistance observed in prostate cancer cells treated with cisplatin. Human LNCaP cells (bearing wild-type p53) and PC-3 cells (lacking p53) were used. The expression levels of protein were determined by western blotting, and the mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cell viability was measured by MTT assay, and the transcriptional effect of small interfering RNA (siRNA) was measured by luciferase reporter gene. We showed that cisplatin treatment increased JNK-1 and JNK-2 activity and expression in both LNCaP and PC-3 cells. In addition, the knockdown of JNK-1 expression by siRNA-JNK-1 or siRNA-JNK-2 significantly impaired the upregulation of AP-1 luciferase reporter gene, but failed to decrease the levels of AP-1 reporter gene expression induced by TPA treatment. Our observations indicate that JNK-1 and JNK-2 may be involved in the chemoresistance observed in prostate cancer cells treated with cisplatin and that blocking the stimulation of Jun kinase (JNK) signaling may be important for regulating the susceptibility to cisplatin of prostate cancer.

PGE2 contributes to TGF-β induced T regulatory cell function in human non-small cell lung cancer

PubMed Central

Baratelli, Felicita; Lee, Jay M; Hazra, Saswati; Lin, Ying; Walser, Tonya C; Schaue, Dorthe; Pak, Peter S; Elashoff, David; Reckamp, Karen; Zhang, Ling; Fishbein, Michael C; Sharma, Sherven; Dubinett, Steven M

2010-01-01

CD4+CD25bright regulatory T cells (Treg) play an important role in cancer-mediated immunosuppression. We and others have previously shown that prostaglandin E2 (PGE2) and transforming growth factor beta (TGF-β) induce CD4+CD25brightFOXP3+Treg. Based on these studies, we investigated the requirement for PGE2 in Treg induction by TGF-β. TGF-β stimulation of human CD4+ T cells induced COX-2-dependent production of PGE2. PGE2-neutralizing antibody treatment significantly reduced the suppressive function of TGF-β-induced Treg (TGF-β-Treg) in vitro. TGF-β concentration measured in the plasma of non-small cell lung cancer (NSCLC) patients directly correlated with the frequency of circulating CD4+CD25brightFOXP3+T cells. Flow cytometry analysis showed increased FOXP3 expression in circulating CD4+CD25+HLA-DR- cells of lung cancer patients compared to control subjects. Immunohistochemical analysis revealed co-expression of TGF-β, COX-2, and FOXP3 in serial sections from resected lung tumor tissues. All together these observations suggest interplay between TGF-β and COX-2 in the induction of Treg activities. Interrupting TGF-β and PGE2 signaling may be important in therapeutic interventions that aim to limit Tregfunction in lung cancer. PMID:20733946

Enteroendocrine K and L cells in healthy and type 2 diabetic individuals.

PubMed

Jorsal, Tina; Rhee, Nicolai A; Pedersen, Jens; Wahlgren, Camilla D; Mortensen, Brynjulf; Jepsen, Sara L; Jelsing, Jacob; Dalbøge, Louise S; Vilmann, Peter; Hassan, Hazem; Hendel, Jakob W; Poulsen, Steen S; Holst, Jens J; Vilsbøll, Tina; Knop, Filip K

2018-02-01

Enteroendocrine K and L cells are pivotal in regulating appetite and glucose homeostasis. Knowledge of their distribution in humans is sparse and it is unknown whether alterations occur in type 2 diabetes. We aimed to evaluate the distribution of enteroendocrine K and L cells and relevant prohormone-processing enzymes (using immunohistochemical staining), and to evaluate the mRNA expression of the corresponding genes along the entire intestinal tract in individuals with type 2 diabetes and healthy participants. In this cross-sectional study, 12 individuals with type 2 diabetes and 12 age- and BMI-matched healthy individuals underwent upper and lower double-balloon enteroscopy with mucosal biopsy retrieval from approximately every 30 cm of the small intestine and from seven specific anatomical locations in the large intestine. Significantly different densities for cells positive for chromogranin A (CgA), glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, peptide YY, prohormone convertase (PC) 1/3 and PC2 were observed along the intestinal tract. The expression of CHGA did not vary along the intestinal tract, but the mRNA expression of GCG, GIP, PYY, PCSK1 and PCSK2 differed along the intestinal tract. Lower counts of CgA-positive and PC1/3-positive cells, respectively, were observed in the small intestine of individuals with type 2 diabetes compared with healthy participants. In individuals with type 2 diabetes compared with healthy participants, the expression of GCG and PYY was greater in the colon, while the expression of GIP and PCSK1 was greater in the small intestine and colon, and the expression of PCSK2 was greater in the small intestine. Our findings provide a detailed description of the distribution of enteroendocrine K and L cells and the expression of their products in the human intestinal tract and demonstrate significant differences between individuals with type 2 diabetes and healthy participants. NCT03044860.

DUAL INHIBITION OF PI3K/AKT AND mTOR SIGNALING IN HUMAN NON-SMALL CELL LUNG CANCER CELLS BY A DIETARY FLAVONOID FISETIN

PubMed Central

Khan, Naghma; Afaq, Farrukh; Khusro, Fatima H.; Adhami, Vaqar Mustafa; Suh, Yewseok; Mukhtar, Hasan

2011-01-01

Lung cancer is one of the most commonly occurring malignancies. It has been reported that mTOR is phosphorylated in lung cancer and its activation was more frequent in tumors with over-expression of PI3K/Akt. Therefore, dual inhibitors of PI3K/Akt and mTOR signaling could be valuable agents for treating lung cancer. In the present study, we show that fisetin, a dietary tetrahydroxyflavone inhibits cell-growth with the concomitant suppression of PI3K/Akt and mTOR signaling in human non-small cell lung cancer (NSCLC) cells. Using autodock 4, we found that fisetin physically interacts with the mTOR complex at two sites. Fisetin treatment was also found to reduce the formation of A549 cell colonies in a dose-dependent manner. Treatment of cells with fisetin caused decrease in the protein expression of PI3K (p85 and p110), inhibition of phosphorylation of Akt, mTOR, p70S6K1, eIF-4E and 4E-BP1. Fisetin-treated cells also exhibited dose-dependent inhibition of the constituents of mTOR signaling complex like Rictor, Raptor, GβL and PRAS40. There was increase in the phosphorylation of AMPKα and decrease in the phosphorylation of TSC2 on treatment of cells with fisetin. We also found that treatment of cells with mTOR inhibitor rapamycin and mTOR-siRNA caused decrease in phosphorylation of mTOR and its target proteins which were further downregulated on treatment with fisetin, suggesting that these effects are mediated in part, through mTOR signaling. Our results show that fisetin suppressed PI3K/Akt and mTOR signaling in NSCLC cells and thus, could be developed as a chemotherapeutic agent against human lung cancer. PMID:21618507

Apoptosis Signal-Regulating Kinase 1 Is Involved in Brain-Derived Neurotrophic Factor (BDNF)-Enhanced Cell Motility and Matrix Metalloproteinase 1 Expression in Human Chondrosarcoma Cells

PubMed Central

Lin, Chih-Yang; Chang, Sunny Li-Yun; Fong, Yi-Chin; Hsu, Chin-Jung; Tang, Chih-Hsin

2013-01-01

Chondrosarcoma is the primary malignancy of bone that is characterized by a potent capacity to invade locally and cause distant metastasis, and is therefore associated with poor prognoses. Chondrosarcoma further shows a predilection for metastasis to the lungs. The brain-derived neurotrophic factor (BDNF) is a small molecule in the neurotrophin family of growth factors that is associated with the disease status and outcome of cancers. However, the effect of BDNF on cell motility in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma cell lines had significantly higher cell motility and BDNF expression compared to normal chondrocytes. We also found that BDNF increased cell motility and expression of matrix metalloproteinase-1 (MMP-1) in human chondrosarcoma cells. BDNF-mediated cell motility and MMP-1 up-regulation were attenuated by Trk inhibitor (K252a), ASK1 inhibitor (thioredoxin), JNK inhibitor (SP600125), and p38 inhibitor (SB203580). Furthermore, BDNF also promoted Sp1 activation. Our results indicate that BDNF enhances the migration and invasion activity of chondrosarcoma cells by increasing MMP-1 expression through a signal transduction pathway that involves the TrkB receptor, ASK1, JNK/p38, and Sp1. BDNF thus represents a promising new target for treating chondrosarcoma metastasis. PMID:23892595

Brief Report: External Beam Radiation Therapy for the Treatment of Human Pluripotent Stem Cell-Derived Teratomas.

PubMed

Lee, Andrew S; Tang, Chad; Hong, Wan Xing; Park, Sujin; Bazalova-Carter, Magdalena; Nelson, Geoff; Sanchez-Freire, Veronica; Bakerman, Isaac; Zhang, Wendy; Neofytou, Evgenios; Connolly, Andrew J; Chan, Charles K; Graves, Edward E; Weissman, Irving L; Nguyen, Patricia K; Wu, Joseph C

2017-08-01

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), have great potential as an unlimited donor source for cell-based therapeutics. The risk of teratoma formation from residual undifferentiated cells, however, remains a critical barrier to the clinical application of these cells. Herein, we describe external beam radiation therapy (EBRT) as an attractive option for the treatment of this iatrogenic growth. We present evidence that EBRT is effective in arresting growth of hESC-derived teratomas in vivo at day 28 post-implantation by using a microCT irradiator capable of targeted treatment in small animals. Within several days of irradiation, teratomas derived from injection of undifferentiated hESCs and hiPSCs demonstrated complete growth arrest lasting several months. In addition, EBRT reduced reseeding potential of teratoma cells during serial transplantation experiments, requiring irradiated teratomas to be seeded at 1 × 10 3 higher doses to form new teratomas. We demonstrate that irradiation induces teratoma cell apoptosis, senescence, and growth arrest, similar to established radiobiology mechanisms. Taken together, these results provide proof of concept for the use of EBRT in the treatment of existing teratomas and highlight a strategy to increase the safety of stem cell-based therapies. Stem Cells 2017;35:1994-2000. © 2017 AlphaMed Press.

HTLV-I Tax-dependent and -independent events associated with immortalization of human primary T lymphocytes

PubMed Central

Bellon, Marcia; Baydoun, Hicham H.; Yao, Yuan

2010-01-01

Human T-cell leukemia virus type I (HTLV-I)–associated malignancies are seen in a small percentage of infected persons. Although in vitro immortalization by HTLV-I virus is very efficient, we report that Tax has poor oncogenic activity in human primary T cells and that immortalization by Tax is rare. Sustained telomerase activity represents one of the oncogenic steps required for Tax-mediated immortalization. Tax expression was required for the growth of primary T cells, but was not sufficient to propel T cells into cell cycle in the absence of exogenous interleukin-2 (IL-2). Tax was sufficient to activate the phosphoinositide-3 kinase (PI3K)/Akt pathway as shown by down regulation of Src homology phosphatase-1 and increased phosphorylation of Akt. We also found disruption of putative tumor suppressors IL-16 and translocated promoter region (TPR) in Tax-immortalized and HTLV-I–transformed cell lines. Our results confirmed previous observations that Tax activates the anaphase-promoting complex. However, Tax did not affect the mitotic spindle checkpoint, which was also functional in HTLV-I–transformed cells. These data provide a better understanding of Tax functions in human T cells, and highlight the limitations of Tax, suggesting that other viral proteins are key to T-cell transformation and development of adult T-cell leukemia. PMID:20093405

Metabolism of the tropine indole-3-carboxylate ICS 205-930 by differentiated rat and human hepatoma cells.

PubMed

Fischer, V; Baldeck, J P; Wiebel, F J

The metabolism of the tropine indole-3-carboxylate ICS 205-930 (ICS), a highly potent and selective antagonist of 5-HT3 receptors, was investigated in continuous cell lines derived from rat or human liver and compared to the in vivo metabolism in rat and human. The well-differentiated rat hepatoma line 2sFou extensively metabolized ICS by hydroxylation of the indole moiety and subsequent conjugation to form the corresponding glucuronides and sulfates. The 2sFou cells also oxidized ICS at the tropinyl moiety to form both N-demethyl and N-oxide derivatives. The relative amount of the various metabolites was dependent on the substrate concentration. Pretreatment of the cells with dexamethasone increased the rate of metabolism for all pathways, while benz[a]anthracene caused an increase in hydroxylation at the indole moiety at the expense of N-oxidation. Phenobarbital pretreatment had no effect on ICS metabolism. The pattern of metabolites formed in 2sFou cells was qualitatively similar to that formed in rat urine. The human hepatoma line HepG2 metabolized ICS only to a small extent. The HepG2 cells failed to form detectable amounts of ICS conjugates found in human urine. The N-oxide-ICS was not found in HepG2 cells or in human urine. Virtually no ICS metabolites were found in human lung adenocarcinoma lines NCI-H358 or NCI-H322. The results suggest that continuous cell lines such as the differentiated rat hepatoma cells 2sFou might be used to mimic the metabolism of xenobiotics in rat and to clarify their complex metabolic pathways.

Spotlight on necitumumab in the treatment of non-small-cell lung carcinoma

PubMed Central

Thakur, Manish K; Wozniak, Antoinette J

2017-01-01

The treatment options for metastatic non-small-cell lung cancer (NSCLC) have expanded dramatically in the last 10 years with the discovery of newer drugs and targeted therapy. Epidermal growth factor receptor (EGFR), when aberrantly activated, promotes cell growth and contributes in various ways to the malignant process. EGFR has become an important therapeutic target in a variety of malignancies. Small-molecule tyrosine kinase inhibitors (TKIs) of EGFR are being used to treat advanced NSCLC and are particularly effective in the presence of EGFR mutations. Monoclonal antibodies have also been developed that block the EGFR at the cell surface and work in conjunction with chemotherapy. Necitumumab is a second-generation fully human IgG1 monoclonal antibody that has shown promise in metastatic NSCLC. The benefit has mostly been restricted to squamous cell lung cancer in the frontline setting. Considering that the survival advantage for these patients was modest, there is a need to discover biomarkers that will predict which patients will likely have the best outcomes. This review focuses on the development and clinical trial experience with necitumumab in NSCLC. PMID:28293124

Selective and cell-active inhibitors of the USP1/UAF1 deubiquitinase complex reverse cisplatin resistance in non-small cell lung cancer cells

PubMed Central

Chen, Junjun; Dexheimer, Thomas S.; Ai, Yongxing; Liang, Qin; Villamil, Mark A.; Inglese, James; Maloney, David J; Jadhav, Ajit; Simeonov, Anton; Zhuang, Zhihao

2012-01-01

Ubiquitin-specific proteases (USPs) have in recent years emerged as a promising therapeutic target class. We identified selective small-molecule inhibitors against a deubiquitinase complex, the human USP1/UAF1, through quantitative high throughput screening (qHTS) of a collection of bioactive molecules. The top inhibitors, pimozide and GW7647, inhibited USP1/UAF1 noncompetitively with a Ki of 0.5 and 0.7 μM respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase and cysteine proteases. The USP1/UAF1 inhibitors act synergistically with cisplatin in inhibiting cisplatin-resistant non-small cell lung cancer (NSCLC) cell proliferation. USP1/UAF1 represents a promising target for drug intervention because of its involvement in translesion synthesis and Fanconi anemia pathway important for normal DNA damage response. Our results support USP1/UAF1 as a potential therapeutic target and provide the first example of targeting the USP/WD40 repeat protein complex for inhibitor discovery. PMID:22118673

Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

PubMed Central

2013-01-01

Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

Cells resembling intraventricular macrophages are present in the subretinal space of human foetal eyes.

PubMed

McMenamin, P G; Loeffler, K U

1990-06-01

The subretinal spaces (SRS) in 17 human foetal eyes were investigated by light microscopy and scanning and transmission electron microscopy. A hitherto undocumented group of pleomorphic cells was detected on the apical surface of the retinal pigment epithelium (RPE) and on the undersurface of the neural retina. These cells formed a regularly spaced array in the peripheral SRS, particularly in the most anterior portion nearest the ciliary body anlage. The morphology of the SRS cells ranged from a small round or ovoid form with a few short basal pseudopodia to an extremely flattened dendritic form. Ultrastructural features, such as large melanophagolysosomes, consistent with a phagocytic function, were observed in some cells. These SRS cells bore remarkable resemblance to epiplexus and supraependymal cells, considered to be the resident population of macrophages on the ventricular surfaces of the brain. This morphological parallelism, together with the anatomically homologous location, is strong evidence that SRS cells represent a normal population of macrophages in the developing human eye. No features consistent with an RPE or neuronal origin were observed. The possible role of these cells as transient phagocytes in the SRS with a possible destiny as retinal microglia is discussed.

HLA Engineering of Human Pluripotent Stem Cells

PubMed Central

Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

2013-01-01

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I–negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8+ T cell responses were reduced in class I–negative cells that had undergone differentiation in embryoid bodies. These B2M−/− ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines. PMID:23629003

HLA engineering of human pluripotent stem cells.

PubMed

Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

2013-06-01

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.

Astaxanthin enhances pemetrexed-induced cytotoxicity by downregulation of thymidylate synthase expression in human lung cancer cells.

PubMed

Liao, Kai-Sheng; Wei, Chia-Li; Chen, Jyh-Cheng; Zheng, Hao-Yu; Chen, Wen-Ching; Wu, Chia-Hung; Wang, Tai-Jing; Peng, Yi-Shuan; Chang, Po-Yuan; Lin, Yun-Wei

2016-11-01

Pemetrexed, a multitargeted antifolate agent, has demonstrated clinical activity in non-small cell lung cancer (NSCLC) cells. Increased expression of thymidylate synthase (TS) is thought to be associated with resistance to pemetrexed. Astaxanthin exhibits a wide range of beneficial effects including anti-cancer and anti-inflammatory properties. In this study, we showed that down-regulating of TS expression in two NSCLC cell lines, human lung adenocarcinoma H1650 and squamous cell carcinoma H1703 cells, with astaxanthin were associated with decreased MKK1/2-ERK1/2 activity. Enforced expression of constitutively active MKK1 (MKK1-CA) vector significantly rescued the decreased TS mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with a MKK1/2 inhibitor (U0126 or PD98059) further decreased the TS expression in astaxanthin-exposed NSCLC cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Combination of pemetrexed and astaxanthin resulted in synergistic enhancing cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-MKK1/2, phopho-ERK1/2, and TS expression. Overexpression of MKK1/2-CA reversed the astaxanthin and pemetrexed-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of MKK1/2-ERK1/2-mediated TS expression by astaxanthin is an important regulator of enhancing the pemetrexed-induced cytotoxicity in NSCLC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

PubMed

Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

1990-01-01

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis.

PubMed

Finka, Andrija; Goloubinoff, Pierre

2013-09-01

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here "the chaperome," which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10% of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.

The physics of cellular synthesis, growth and division

NASA Technical Reports Server (NTRS)

Pollard, E. C.

1974-01-01

Three areas of research in NASA'S University Program are described. Primitive terrestrial living cells were studied as a guide to the kind of cells to look for in extraterrestrial life. Experiments in zero gravity conditions are described with emphasis upon effects on small organisms. The effects of ionizing radiation on cells are studied so that it will be possible to predict dosages which can be tolerated by humans with no permanent damage.

WAVE2- and microtubule-dependent formation of long protrusions and invasion of cancer cells cultured on three-dimensional extracellular matrices.

PubMed

Kikuchi, Keiji; Takahashi, Kazuhide

2008-11-01

Invadopodia, small protrusions formed at ventral membranes of several types of invasive cancer cells upon contact with the extracellular matrix (ECM), are implicated in cell invasion; however, the relationship between invadopodia formation and cell invasion through the ECM is still unknown. To correlate the formation of membrane protrusions and cell invasion, a three-dimensional (3-D) gel culture system with native collagen type-I matrix overlaid with a thin basement membrane equivalent (Matrigel) was made. Human breast cancer cell line MDA-MB-231 formed long protrusions in addition to small protrusions reminiscent of invadopodia and migrated into the collagen layer. Comparative analyses with other cancer cell lines indicate that cellular ability to form long protrusions, but not small protrusions or invadopodia, correlates with cellular invasiveness in the 3-D culture. Some of the long protrusions in MDA-MB-231 cells appeared to extend from the adherence membrane, implying that they are derived from small protrusions. The formation of long protrusions and invasion, as well as the formation of invadopodia, required WAVE2 in MDA-MB-231 cells. Accumulation of tubulin was observed in long protrusions but not in invadopodia. Correspondingly, a microtubule-stabilizing agent, paclitaxel, suppressed the formation of long protrusions and invasion, but not the formation of invadopodia, in MDA-MB-231 cells. These results suggest that long protrusions formed in a WAVE2- and microtubule-dependent manner may identify the cells at the later stage of invasion, possibly after the formation of invadopodia in the 3-D cultures.

Interaction of Human Enterochromaffin Cells with Human Enteric Adenovirus 41 Leads to Serotonin Release and Subsequent Activation of Enteric Glia Cells.

PubMed

Westerberg, Sonja; Hagbom, Marie; Rajan, Anandi; Loitto, Vesa; Persson, B David; Allard, Annika; Nordgren, Johan; Sharma, Sumit; Magnusson, Karl-Eric; Arnberg, Niklas; Svensson, Lennart

2018-04-01

Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells. IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41 infection on human enterochromaffin cells and found it stimulates serotonin secretion in the cells, which is involved in regulation of intestinal secretion and gut motility and can also activate enteric glia cells, which are found in close proximity to enterochromaffin cells in vivo This disruption of gut barrier homeostasis as maintained by these cells following human adenovirus 41 infection might be a mechanism in enteric adenovirus pathogenesis in humans and could indicate a possible serotonin-dependent cross talk between human adenovirus 41, enterochromaffin cells, and enteric glia cells. Copyright © 2018 American Society for Microbiology.

Nicotine promotes vascular endothelial growth factor secretion by human trophoblast cells under hypoxic conditions and improves the proliferation and tube formation capacity of human umbilical endothelial cells.

PubMed

Zhao, Hongbo; Wu, Lanxiang; Wang, Yahui; Zhou, Jiayi; Li, Ruixia; Zhou, Jiabing; Wang, Zehua; Xu, Congjian

2017-04-01

Pre-eclampsia, characterized as defective uteroplacental vascularization, remains the major cause of maternal and fetal mortality and morbidity. Previous epidemiological studies demonstrated that cigarette smoking reduced the risk of pre-eclampsia. However, the molecular mechanism remains elusive. In the present study, it is demonstrated that a low dose of nicotine decreased soluble vascular endothelial growth factor receptor 1 (sFlt1) secretion in human trophoblast cells under hypoxic conditions. Nicotine was then observed to promote vascular endothelial growth factor (VEGF) secretion by reducing sFlt1 secretion and increasing VEGF mRNA transcription. Further data showed that nicotine enhanced hypoxia-mediated hypoxia-inducible factor-1α (HIF-1α) expression and HIF-1α small interfering RNA abrogated nicotine-induced VEGF secretion, indicating that HIF-1α may be responsible for nicotine-mediated VEGF transcription under hypoxic conditions. Moreover, conditioned medium from human trophoblast cells treated with nicotine under hypoxic conditions promoted the proliferation and tube formation capacity of human umbilical endothelial cells (HUVEC) by promoting VEGF secretion. These findings indicate that nicotine may promote VEGF secretion in human trophoblast cells under hypoxic conditions by reducing sFlt1 secretion and up-regulating VEGF transcription and improve the proliferation and tube formation of HUVEC cells, which may contribute to elucidate the protective effect of cigarette smoking against pre-eclampsia. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Effects of human interleukin-18 and interleukin-12 treatment on human lymphocyte engraftment in NOD-scid mouse

PubMed Central

Senpuku, Hidenobu; Asano, Toshihiko; Matin, Khairul; Salam, M Abdus; Tsuha, Yuzo; Horibata, Shigeo; Shimazu, Yoshihito; Soeno, Yuichi; Aoba, Takaaki; Sata, Tetsutaro; Hanada, Nobuhiro; Honda, Mitsuo

2002-01-01

NOD/LtSz-prkdcscid/prkdcscid (non-obese diabetic-severe combine immunodeficiency; NOD-scid) mice grafted with human peripheral blood lymphoid cells have been used as an in vivo humanized mouse model in various studies. However, cytotoxic human T cells are induced in this model during immune responses, which gives misleading results. To assist in grafting of human lymphocytes without the induction of cytotoxic human T cells, we investigated the effects of T helper type 1 (Th1) and Th2 cytokines on human lymphocyte grafting and migration, as well as the production of immunoglobulin deposited in glomeruli and human immunodeficiency virus-1 (HIV-1) infection using NOD-scid mice. Administration of interleukin-18 (IL-18) and IL-12 enhanced the grafting of human CD4+ and CD8+ T cells in the mice, whereas co-administration prevented grafting due to interferon-γ-dependent apoptosis. Immunoglobulin A (IgA) deposits were observed in mice treated with IL-18 alone, but not in those given phosphate-buffered saline, IL-12 alone, or IL-18 + IL-12. A high rate of HIV infection was also observed in the IL-18-treated group. Together, these results indicate that IL-18 may be effective for the grafting and migration of CD4+ and CD8+ T cells, except for the induction of apoptosis and regulation of class-switching IgA. IL-18-administered NOD-scid mice provide a useful small humanized model for the study of HIV infection and IgA nephropathy. PMID:12383203

Analysis of T cell-replacing factor-like activity: potent induction of T helper activity for human B cells by residual concanavalin A and interleukin 2.

PubMed

Sauerwein, R W; Van der Meer, W G; Aarden, L A

1987-08-01

At least two factors with the capacity to induce IgM synthesis in human B cells were found to be present in the 15-20-kDa fraction of the supernatant of mononuclear cells activated with concanavalin A (Con A) and phorbol ester. Previously, it has been shown (Sauerwein, R. W. et al., Eur. J. Immunol. 1985. 15: 611) that interleukin 2 (IL2) in this material is able to induce T cell-dependent IgM secretion in normal B cells. Evidence was obtained for the presence of another factor distinct from IL2 that could replace T cells in the induction of B cell differentiation. We have analyzed this factor with the use of a neoplastic B cell population of prolymphocytic origin that was functionally nonresponsive to IL2. T cell-replacing factor (TRF)-like activity and IL2 could be separated by ion-exchange chromatography, although a small amount of IL2 was recovered in the TRF fractions. This small amount of IL2 was found to be crucial for the observed TRF activity. Moreover, a substantial amount of monomeric Con A was detected in the TRF preparation. Our studies show that Con A in the presence of IL2 can act as a potent inducer of helper function in lower numbers of T cells for normal and neoplastic B cells. Functional assays for T cell contamination in B cell suspensions are therefore of limited value because they are determined by the efficiency of the stimulating signal. Particularly in those B cell factor preparations, obtained from mitogen-activated T cells with an obligatory or unidentified role of IL2, the possible effect of a contaminating mitogen must be considered.

IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer.

PubMed

Kryczek, Ilona; Wu, Ke; Zhao, Ende; Wei, Shuang; Vatan, Linhua; Szeliga, Wojciech; Huang, Emina; Greenson, Joel; Chang, Alfred; Roliński, Jacek; Radwan, Piotr; Fang, Jingyuan; Wang, Guobin; Zou, Weiping

2011-04-01

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.

Discovery and Testing of Ricin Therapeutics

DTIC Science & Technology

2012-06-01

variants lung disease ER degradation Fabri disease α -D-galactosidase neurological disease ER degradation Oculocutaneous albinism tyrosinase...against other pathogens and human diseases . 15. SUBJECT TERMS Ricin toxin, dislocation, small weight compounds, chemical inhibitors, high-content screen...the U.S. Departments of Health and Human Services (HHS) and Centers for Disease Control and Prevention (CDC). The toxin’s ability to kill cells and

Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle

PubMed Central

Yim, Peter D.; Gallos, George; Perez-zoghbi, Jose F.; Trice, Jacquelyn; Zhang, Yi; Siviski, Matthew; Sonett, Joshua; Emala, Charles W.

2014-01-01

Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K+ channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders. PMID:24662476

A synthetic cryptochrome inhibitor induces anti-proliferative effects and increases chemosensitivity in human breast cancer cells

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

Chun, Sung Kook; Department of Biological Sciences, Seoul National University, Seoul, 151-747; Department of Brain & Cognitive Sciences, Seoul National University, Seoul, 151-747

Disruption of circadian rhythm is a major cause of breast cancer in humans. Cryptochrome (CRY), a circadian transcription factor, is a risk factor for initiation of breast cancer, and it is differentially expressed between normal and breast cancer tissues. Here, we evaluated the anti-proliferative and pro-apoptotic activity of KS15, a recently discovered small-molecule inhibitor of CRY, in human breast cancer cells. First, we investigated whether KS15 treatment could promote E-box-mediated transcription by inhibiting the activity of CRY in MCF-7 human breast cancer cells. Protein and mRNA levels of regulators of cell cycle and apoptosis, as well as core clock genes,more » were differentially modulated in response to KS15. Next, we investigated whether KS15 could inhibit proliferation and increase sensitivity to anti-tumor drugs in MCF-7 cells. We found that KS15 decreased the speed of cell growth and increased the chemosensitivity of MCF-7 cells to doxorubicin and tamoxifen, but had no effect on MCF-10A cells. These findings suggested that pharmacological inhibition of CRY by KS15 exerts an anti-proliferative effect and increases sensitivity to anti-tumor drugs in a specific type of breast cancer. - Highlights: • Cryptochrome inhibitor (KS15) has anti-tumor activity to human breast cancer cells. • KS15 induces differential changes in cell cycle regulators and pro-apoptotic genes. • KS15 inhibits MCF-7 cell growth and enhances susceptibility to anti-tumor drugs.« less

The Expanding Family of Virophages.

PubMed

Bekliz, Meriem; Colson, Philippe; La Scola, Bernard

2016-11-23

Virophages replicate with giant viruses in the same eukaryotic cells. They are a major component of the specific mobilome of mimiviruses. Since their discovery in 2008, five other representatives have been isolated, 18 new genomes have been described, two of which being nearly completely sequenced, and they have been classified in a new viral family, Lavidaviridae . Virophages are small viruses with approximately 35-74 nm large icosahedral capsids and 17-29 kbp large double-stranded DNA genomes with 16-34 genes, among which a very small set is shared with giant viruses. Virophages have been isolated or detected in various locations and in a broad range of habitats worldwide, including the deep ocean and inland. Humans, therefore, could be commonly exposed to virophages, although currently limited evidence exists of their presence in humans based on serology and metagenomics. The distribution of virophages, the consequences of their infection and the interactions with their giant viral hosts within eukaryotic cells deserve further research.

Characterization of side population in thyroid cancer cell lines: cancer stem-like cells are enriched partly but not exclusively.

PubMed

Mitsutake, Norisato; Iwao, Atsuhiko; Nagai, Kazuhiro; Namba, Hiroyuki; Ohtsuru, Akira; Saenko, Vladimir; Yamashita, Shunichi

2007-04-01

There is increasing evidence that cancers contain their own stem-like cells called cancer stem cells (CSCs). A small subset of cells, termed side population (SP), has been identified using flow cytometric analysis. The SP cells have the ability to exclude the DNA binding dye, Hoechst33342, and are highly enriched for stem cells in many kinds of normal tissues. Because CSCs are thought to be drug resistant, SP cells in cancers might contain CSCs. We initially examined the presence of SP cells in several human thyroid cancer cell lines. A small percentage of SP cells were found in ARO (0.25%), FRO (0.1%), NPA (0.06%), and WRO (0.02%) cells but not TPC1 cells. After sorting, the SP cells generated both SP and non-SP cells in culture. The clonogenic ability of SP cells was significantly higher than that of non-SP cells. Moreover, the SP prevalence was dependent on cell density in culture, suggesting that SP cells preferentially survived at lower cell density. Microarray experiment revealed differential gene expression profile between SP and non-SP cells, and several genes related to stemness were up-regulated. However, non-SP population also contained cells that were tumorigenic in nude mice, and non-SP cells generated a small number of SP cells. These results suggest that cancer stem-like cells are partly, but not exclusively, enriched in SP population. Clarifying the key tumorigenic population might contribute to the establishment of a novel therapy for thyroid cancer.

A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells

PubMed Central

Chaikind, Brian; Bessen, Jeffrey L.; Thompson, David B.; Hu, Johnny H.; Liu, David R.

2016-01-01

We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases. PMID:27515511