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Sample records for genes radical-induced cell

  1. The paralogous genes RADICAL-INDUCED CELL DEATH1 and SIMILAR TO RCD ONE1 have partially redundant functions during Arabidopsis development.

    Science.gov (United States)

    Teotia, Sachin; Lamb, Rebecca S

    2009-09-01

    RADICAL-INDUCED CELL DEATH1 (RCD1) and SIMILAR TO RCD ONE1 (SRO1) are the only two proteins encoded in the Arabidopsis (Arabidopsis thaliana) genome containing both a putative poly(ADP-ribose) polymerase catalytic domain and a WWE protein-protein interaction domain, although similar proteins have been found in other eukaryotes. Poly(ADP-ribose) polymerases mediate the attachment of ADP-ribose units from donor NAD(+) molecules to target proteins and have been implicated in a number of processes, including DNA repair, apoptosis, transcription, and chromatin remodeling. We have isolated mutants in both RCD1 and SRO1, rcd1-3 and sro1-1, respectively. rcd1-3 plants display phenotypic defects as reported for previously isolated alleles, most notably reduced stature. In addition, rcd1-3 mutants display a number of additional developmental defects in root architecture and maintenance of reproductive development. While single mutant sro1-1 plants are relatively normal, loss of a single dose of SRO1 in the rcd1-3 background increases the severity of several developmental defects, implying that these genes do share some functions. However, rcd1-3 and sro1-1 mutants behave differently in several developmental events and abiotic stress responses, suggesting that they also have distinct functions. Remarkably, rcd1-3; sro1-1 double mutants display severe defects in embryogenesis and postembryonic development. This study shows that RCD1 and SRO1 are at least partially redundant and that they are essential genes for plant development.

  2. Inhibition of free radical induced oxidative hemolysis of red blood cells by green tea polyphenols

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The in vitro oxidative hemolysis of human red blood cells (RBC) was used as a model to study the free radical induced damage of biological membranes and the inhibitory effect of natural antioxidants. The hemolysis was induced by a water-soluble free radical initiator 2,2′-azo(2- asmidinopropane)dihydrochloride (AAPH) and inhibited by the principal polyphenolic components extracted from green tea leaves, i.e. (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicat- echin gallate (ECG), (-)-epigallocatechin gallate (EGCG) and gallic acid (GA). Addition of AAPH at 37°C caused fast hemolysis after a short period of inhibition period, while addition of the green tea polyphenols efficiently suppressed the hemolysis in the activity sequence of EGCG>EGC>ECG≈EC>GA, demonstrating that these green tea polyphenols are effective antioxidants which could protect biological membranes from free radical induced oxidative damage.

  3. Antioxidative Effect of Schiff Bases with o-Hydroxybenzylidene Groupon Free Radical Induced Hemolysis of Human Red Blood Cell

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The hemolysis of human red blood cells was initiated by a water-soluble free radical initiator, 2,2′-azobis-(2-amidinopropane hydrochloride)(AAPH), at 37 ℃ in phosphate buffered saline(pH=7.4). The respective addition of 1-[N-(o-hydroxybenzylidene)amino]tetradecane(TDCA), 1,2-di[N-(o-hydroxybenzylidene)amino]cyclohexane(DACH), 4-[N-(o-hydroxybenzylidene)amino]benzoic acid(PABA), 4-nitro[N-(o-hydroxybenzylidene)]aniline(APNA) or N-(o-hydroxybenzylidene)aniline(APA) can all prolong the inhibition period of hemolysis, indicating that the above Schiff bases play an antioxidative role in free radical induced hemolysis. It can be concluded that Schiff base with an alkyl group or a conjugated system in the molecule protect red blood cells against free radical induced hemolysis efficiently. This information may be useful for antioxidant drug design.

  4. Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses.

    Science.gov (United States)

    Ahlfors, Reetta; Lång, Saara; Overmyer, Kirk; Jaspers, Pinja; Brosché, Mikael; Tauriainen, Airi; Kollist, Hannes; Tuominen, Hannele; Belles-Boix, Enric; Piippo, Mirva; Inzé, Dirk; Palva, E Tapio; Kangasjärvi, Jaakko

    2004-07-01

    Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain-containing subfamily of the WWE protein-protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.

  5. Protective Effects of Resveratrol and its Analogues against Free Radical-Induced Oxidative Hemolysis of Red Blood Cells

    Institute of Scientific and Technical Information of China (English)

    FANG,Jian-Guo(房建国); LU,Man(陆曼); MA,Lan-Ping(马兰萍); YANG,Li(杨立); WU,Long-Min(吴隆民); LIU,Zhong-Li(刘中立)

    2002-01-01

    The in vitro oxidative hemolysis of human red blood cells ( RBCs) was used as a model to study the free radical-induced damage of biological membranes and the protective effect of resveratroi (3,5,4′-trihydroxy-trans-stilbene, 1) and its analogues, i. e., 4-hydroxy-trans-stilbene(2), 3, 5-dihydroxytrans-stilbene (3), 3,4-dihydroxy-trans-stilbene (4), 4,4′-dihydroxy-trans-stilbene (5) and 2, 4, 4′-trihydroxy-trans-stilbene (6). The hemolysis of RBCs was induced by a water-soluble free radical initiator 2, 2′-azobis ( 2-amidinopropane hydrochloride) (AAPH). It was found that addition of AAPH at 37 ℃ to the suspension of RBCs caused fast hemolysis after a short period of inhibition period, and addition of 1-6 significantly suppressed the hemolysis. Compound 4 which bears an ortho-dihydroxyl functionality showed nuch more effective anti-hemolysis activity than that of resveratrol and the other analogues.

  6. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  7. Senescence-associated Barley NAC (NAM, ATAF1,2, CUC) Transcription Factor Interacts with Radical-induced Cell Death 1 through a Disordered Regulatory Domain

    DEFF Research Database (Denmark)

    Kjærsgaard, Trine; Jensen, Michael Krogh; Wagner, Michael

    2011-01-01

    as a transcriptional activator suggesting that an involvement of HvNAC013 and HvNAC005 in senescence will be different. HvNAC013 interacted with barley radical-induced cell death 1 (RCD1) via the very C-terminal part of its TRD, outside of the region containing the LP motif. No significant secondary structure...... was induced in the HvNAC013 TRD upon interaction with RCD1. RCD1 also interacted with regions dominated by intrinsic disorder in TFs of the MYB and basic helix-loop-helix families. We propose that RCD1 is a regulatory protein capable of interacting with many different TFs by exploiting their intrinsic...

  8. Structures and short linear motif of disordered transcription factor regions provide clues to the interactome of the cellular hub radical-induced cell death1

    DEFF Research Database (Denmark)

    O'Shea, Charlotte; Staby, Lasse; Bendsen, Sidsel Krogh

    2017-01-01

    point to larger networks of interactions, such as with proteins that serve as hubs for essential cellular functions. The stress-associated plant protein Radical-Induced Cell Death1 (RCD1) is one such hub, interacting with many transcription factors via their flexible IDRs. To identify the SLiM bound...... by RCD1, we analyzed the IDRs in three protein partners - DREB2A, ANAC013, and ANAC046 - considering parameters such as disorder, context, charges, and pI. Using a combined bioinformatics and experimental approach, we have identified the bipartite RCD1-binding SLiM as [DE]-x(1,2)-[YF]-x(1,4)-[DE......]-L, with essential contributions from conserved aromatic, acidic, and leucine residues. Detailed thermodynamic analysis revealed both favorable and unfavorable contributions from the IDRs surrounding the SLiM to the interactions with RCD1, and the SLiM affinities ranged from low nanomolar to 50 times higher Kd...

  9. Lipid Peroxidation-Mediated Telomere Shortening in Hydroxyl Radical-Induced Apoptosis in HeLa Cells

    Institute of Scientific and Technical Information of China (English)

    任建国; 陈晶; 戴尧仁

    2001-01-01

    Many anti-cancer drugs have been found to trigger apoptosis in tumor cells through the production of reactive oxygen species (ROS) including hydroxyl radicals (@ OH) regardless of chemical types. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However, little is known about the linkage between ROS (such as @ OH) and telomerase/telomere. The focus of this investigation was to examine the possible pathway of the apoptosis induced by @ OH production via Fe2+ and H2O2. Results of the present study demonstrated that after exposure of HeLa cells to Fe2+-H2O2 system, an increase in lipid peroxidation and reduction of GSH was observed. These events proceeded and triggered apoptosis, resulting in DNA fragmentation. More interestingly, we did not observe any changes of telomerase activity. However, the telomere length in apoptotic cells shortened significantly. We also found that GSH rescued @ OH-induced HeLa cell death and prevened telomere shortening, and that 3,3'-diethyoxadicarbocyanine (DODCB), a telomerase inhibitor, increased susceptibility of HeLa cells to @ OH-induced apoptosis. Our results suggest that @ OH-induced telomere shortening is not through telomerase inhibition but possibly a direct effect of @ OH on telomeres themselves via lipid peroxidation.

  10. Protective Effect of Sinapine against Hydroxyl Radical-Induced Damage to Mesenchymal Stem Cells and Possible Mechanisms.

    Science.gov (United States)

    Li, Xican; Han, Lu; Li, Yunrong; Zhang, Jing; Chen, Jiemin; Lu, Wenbiao; Zhao, Xiaojun; Lai, Yingtao; Chen, Dongfeng; Wei, Gang

    2016-01-01

    As a phenolic alkaloid occurring in Cruciferous plants, sinapine was observed to protect mesenchymal stem cells (MSCs) against ·OH-induced damage in this study. It was also found to prevent DNA from damage, to scavenge various free radicals (·OH, ·O2(-), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS)(+·), and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·)), and to reduce Cu(2+) to Cu(+). To further explore the mechanism, the end-product of sinapine reaction with DPPH· was determined using HPLC-electrospray ionization (ESI)-MS/MS and HPLC-diode array detector (DAD). Four molecular ion peaks (m/z 701, 702, 703, and 351) in HPLC-ESI-MS/MS analysis indicated a radical adduct formation (RAF) pathway; while a bathochromic shift (λ(max) 334→475 nm) in HPLC-DAD indicated the formation of quinone as the oxidized product of the phenolic -OH group. Based on these results, it may be concluded that, (i) sinapine can effectively protect against ·OH-induced damage to DNA and MSCs; such protective effect may provide evidence for a potential role for sinapine in MSC transplantation therapy, and be responsible for the beneficial effects of Cruciferous plants. (ii) The possible mechanism for sinapine to protect against ·OH-induced oxidative damage is radical-scavenging, which is thought to be via hydrogen atom (H·) transfer (HAT) (or sequential electron (e) proton transfer (SEPT))→RAF pathways.

  11. Influence of Cocoa Flavanols and Procyanidins on Free Radical-induced Human Erythrocyte Hemolysis

    Directory of Open Access Journals (Sweden)

    Qin Yan Zhu

    2005-01-01

    Full Text Available Cocoa can be a rich source of antioxidants including the flavan-3-ols, epicatechin and catechin, and their oligomers (procyanidins. While these flavonoids have been reported to reduce the rate of free radical-induced erythrocyte hemolysis in experimental animal models, little is known about their effect on human erythrocyte hemolysis. The major objective of this work was to study the effect of a flavonoid-rich cocoa beverage on the resistance of human erythrocytes to oxidative stress. A second objective was to assess the effects of select purified cocoa flavonoids, epicatechin, catechin, the procyanidin Dimer B2 and one of its major metabolites, 3ʹ-O-methyl epicatechin, on free radical-induced erythrocyte hemolysis in vitro. Peripheral blood was obtained from 8 healthy subjects before and 1, 2, 4 and 8 h after consuming a flavonoid-rich cocoa beverage that provided 0.25 g/kg body weight (BW, 0.375 or 0.50 g/kg BW of cocoa. Plasma flavanol and dimer concentrations were determined for each subject. Erythrocyte hemolysis was evaluated using a controlled peroxidation reaction. Epicatechin, catechin, 3ʹ-O-methyl epicatechin and (--epicatechin-(4β > 8epicatechin (Dimer B2 were detected in the plasma within 1 h after the consumption of the beverage. The susceptibility of erythrocytes to hemolysis was reduced significantly following the consumption of the beverages. The duration of the lag time, which reflects the capacity of cells to buffer free radicals, was increased. Consistent with the above, the purified flavonoids, epicatechin, catechin, Dimer B2 and the metabolite 3ʹ-O-methyl epicatechin, exhibited dose-dependent protection against AAPH-induced erythrocyte hemolysis at concentrations ranging from 2.5 to 20 μM. Erythrocytes from subjects consuming flavonoid-rich cocoa show reduced susceptibility to free radical-induced hemolysis (p < 0.05.

  12. Overexpression of the RADICAL-INDUCED CELL DEATH1 (RCD1) Gene of Arabidopsis Causes Weak rcd1 Phenotype with Compromised Oxidative-Stress Responses

    OpenAIRE

    Fujibe, Takahiro; Saji, Hikaru; Watahiki, Masaaki K; Yamamoto, Kotaro T

    2006-01-01

    rcd1 is a mutant of Arabidopsis thaliana that is more resistant to methyl viologen, but more sensitive to ozone than the wild type. rcd1-2 is caused by a single nucleotide substitution that results in a premature stop codon at Trp-332. The rcd1-2 mRNA level does not change significantly with the mutation. Since overexpression of rcd1-1 cDNA has been shown to bring about an rcd1-like phenotype, we created and examined the overexpression lines of RCD1 by the use of the cauliflower mosaic virus ...

  13. Ex-vivo and in vitro protective effects of kolaviron against oxygen-derived radical-induced DNA damage and oxidative stress in human lymphocytes and rat liver cells

    DEFF Research Database (Denmark)

    Farombi, E.O.; Moller, P.; Dragsted, L.O.

    2004-01-01

    mumol/L increased the levels of DNA strand breaks and oxidized purine (formamido-pyrimidine glycosylase (FPG) and pyrimidine (endonuclease III (ENDO III) sites) bases in both human lymphocytes and rat liver cells using alkaline single cell gel electrophoresis (the comet assay). Kolaviron was protective...

  14. Hydroxyl Radical Induced Apoptosis Is Closely Related to Changes in Cellular Energy/Redox Metabolism

    Institute of Scientific and Technical Information of China (English)

    贺雨虹; 陈晶; 任建国; 隋森芳; 蔡国平

    2003-01-01

    Reactive oxygen species (ROS), including the hydroxyl radical (·OH), are known to be potential modulators of apoptosis.However, the biochemical mechanisms underlying apoptosis induced by·OH, namely how the radical induces a cell to die, are poorly understood.The present work highlights the changes of the energy/redox status during apoptosis by exogenous· OH-treatment.HeLa cells were induced to undergo typical apoptosis by·OH generated directly via the Fe2+-mediated Fenton reaction.The thermodynamics study indicated that the· OH-treatment increased the cellular heat output in the first hours, and then the cellular thermodynamics shifted to endothermic.The data demonstrates that the mitochondria are actively involved in· OH-treatment induced apoptosis, with the cellular oxygen consumption rapidly decreasing after the·OH-treatment for only 0.5 h.But DNA fragmentation, which is the major characteristic of apoptosis, took place 16 h after · OH-treatment.The results suggest that alteration of the energy/redox metabolism and the energy/redox status may be the primary causes among the early events of· OH-induced apoptosis.

  15. Synthesis and characterization of partially fluorinated poly(acryl) ionomers for polymer electrolyte membrane fuel cells and ESR-spectroscopic investigation of the radically induced degradation of model compounds; Synthese und Charakterisierung teilfluorierter Poly(acryl)-Ionomere als Polymerelektrolytmembranen fuer Brennstoffzellen und ESR-spektroskopische Untersuchung der radikalinduzierten Degradation von Modellverbindungen

    Energy Technology Data Exchange (ETDEWEB)

    Schoenberger, Frank

    2008-07-09

    this work deals with the EPR-spectroscopic investigation of radically induced degradation reactions of model compounds which represent structural units of poly(aryl) ionomers prepared in the first part of this work. These model compounds are exposed to hydroxyl and hydroperoxyl radicals in a flow cell, which are generated directly by photolysis of hydrogen peroxide in the cavity of an ESR spectrometer. By using this experimental setup different parameters (such as concentration of hydroxyl radicals, monomer concentration, flow rate, and pH value) are varied systematically and their influences in terms of the observed product formation of the aromatic model compounds with the hydroxyl radicals are estimated. Conclusions in terms of possible radical reactions of the poly(aryl) ionomer can be drawn from these investigations and information of avoidable structural features (e.g. type of the end groups of the ionomers) and avoidable conditions (e.g. inhomogeneities of pH values in the membrane) are obtained. (orig.)

  16. Endomorphins, endogenous opioid peptides, provide antioxidant defense in the brain against free radical-induced damage.

    Science.gov (United States)

    Lin, Xin; Yang, Ding-Jian; Cai, Wen-Qing; Zhao, Qian-Yu; Gao, Yan-Feng; Chen, Qiang; Wang, Rui

    2003-11-20

    Oxidative stress has been considered to be a major cause of cellular injuries in a variety of chronic health problems, such as carcinogenesis and neurodegenerative disorders. The brain appears to be more susceptible to oxidative damage than other organs. Therefore, the existence of antioxidants may be essential in brain protective systems. The antioxidative and free radical scavenging effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, have been investigated in vitro. The oxidative damage was initiated by a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrocholoride) (AAPH) and hydrogen peroxide (H2O2). The linoleic acid peroxidation, DNA and protein damage were monitored by formation of hydroperoxides, by plasmid pBR 322 DNA nicking assay and single-cell alkaline electrophoresis, and by SDS-polyacrylamide gel electrophoresis. Endomorphins can inhibit lipid peroxidation, DNA strand breakage, and protein fragmentation induced by free radical. Endomorphins also reacted with galvinoxyl radicals in homogeneous solution, and the pseudo-first-order rate constants were determined spectrophotometrically by following the disappearance of galvinoxyl radicals. In all assay systems, EM1 was more potent than EM2 and GSH, a major intracellular water-soluble antioxidant. We propose that endomorphins are one of the protective systems against free radical-induced damage in the brain.

  17. Clock Genes in Glia Cells

    Science.gov (United States)

    Chi-Castañeda, Donají

    2016-01-01

    Circadian rhythms are periodic patterns in biological processes that allow the organisms to anticipate changes in the environment. These rhythms are driven by the suprachiasmatic nucleus (SCN), the master circadian clock in vertebrates. At a molecular level, circadian rhythms are regulated by the so-called clock genes, which oscillate in a periodic manner. The protein products of clock genes are transcription factors that control their own and other genes’ transcription, collectively known as “clock-controlled genes.” Several brain regions other than the SCN express circadian rhythms of clock genes, including the amygdala, the olfactory bulb, the retina, and the cerebellum. Glia cells in these structures are expected to participate in rhythmicity. However, only certain types of glia cells may be called “glial clocks,” since they express PER-based circadian oscillators, which depend of the SCN for their synchronization. This contribution summarizes the current information about clock genes in glia cells, their plausible role as oscillators and their medical implications. PMID:27666286

  18. DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions.

    Science.gov (United States)

    Satoh, M S; Jones, C J; Wood, R D; Lindahl, T

    1993-07-01

    Plasmid DNA was gamma-irradiated or treated with H2O2 in the presence of Cu2+ to generate oxygen free radical-induced lesions. Open circular DNA molecules were removed by ethidium bromide/CsCl density gradient centrifugation. The closed circular DNA fraction was treated with the Escherichia coli reagent enzymes endonuclease III (Nth protein) and Fpg protein. This treatment converted DNA molecules containing the major base lesions pyrimidine hydrates and 8-hydroxyguanine to a nicked form. Remaining closed circular DNA containing other oxygen radical-induced base lesions was used as a substrate for nucleotide excision-repair in a cell-free system. Extracts from normal human cells, but not extracts from xeroderma pigmentosum cells, catalyzed repair synthesis in this DNA. The repair defect in the latter extracts could be specifically corrected by in vitro complementation. The data suggest that accumulation of endogenous oxidative damage in cellular DNA from xeroderma pigmentosum patients contributes to the increased frequency of internal cancers and the neural degeneration occurring in serious cases of the syndrome.

  19. Protective effect of saponins from Argania spinosa against free radical-induced oxidative haemolysis.

    Science.gov (United States)

    Amzal, H; Alaoui, K; Tok, S; Errachidi, A; Charof, R; Cherrah, Y; Benjouad, A

    2008-07-01

    Saponins from Argania spinosa at a non-haemolytic concentration diminish by 53.2% erythrocyte haemolysis induced by free radicals. 2 mM aspirin and acetaminophen diminish by 75% and 68% , respectively, erythrocyte haemolysis induced by free radicals, while 0.3 microM vitamin E shows no significant antioxidant activity. Interestingly, a combination of 1 mg/l of A. spinosa saponins and vitamin E at 0.3 microM resulted in a 68% level of protection against free radical-induced erythrocyte haemolysis, which may suggest that A. spinosa saponins enhance the antioxidant effect of vitamin E. In contrast, no synergic effect was observed for acetaminophen (2 mM) when in combination with vitamin E (0.3 microM). These results demonstrate the antioxidant properties of saponins from A. spinosa and their ability to potentate the antioxidant effect of vitamin E.

  20. Protective effects of glucosamine hydrochloride against free radical-induced erythrocytes damage.

    Science.gov (United States)

    Jamialahmadi, Khadijeh; Arasteh, Omid; Matbou Riahi, Maryam; Mehri, Soghra; Riahi-Zanjani, Bamdad; Karimi, Gholamreza

    2014-07-01

    Glucosamine (GlcN) is an important precursor in the biochemical synthesis of glycosylated proteins and lipids in human body. It gains importance because of its contribution to human health and its multiple biological and therapeutic effects. In this study, the in vitro oxidative hemolysis of rat erythrocyte was used as a model to study the potential protective effect of glucosamine hydrochloride against free radical-induced damage of biological membranes. Glucosamine hydrochloride exhibited dose-dependent DPPH antioxidant activity. Oxidative hemolysis and lipid/protein peroxidation of erythrocytes induced by a water-soluble free radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were significantly suppressed by GlcN in a time and dose dependent manner. GlcN also prevented the depletion of cytosolic antioxidant glutathione (GSH) in erythrocytes. These results indicated that glucosamine hydrochloride efficiently protected erythrocytes against free radicals and it could be recommended as a pharmaceutical supplement to alleviate oxidative stress.

  1. UV-Induced Adenine Radicals Induced in DNA A-Tracts: Spectral and Dynamical Characterization.

    Science.gov (United States)

    Banyasz, Akos; Ketola, Tiia-Maaria; Muñoz-Losa, Aurora; Rishi, Sunny; Adhikary, Amitava; Sevilla, Michael D; Martinez-Fernandez, Lara; Improta, Roberto; Markovitsi, Dimitra

    2016-10-06

    Adenyl radicals generated in DNA single and double strands, (dA)20 and (dA)20·(dT)20, by one- and two-photon ionization by 266 nm laser pulses decay at 600 nm with half-times of 1.0 ± 0.1 and 4 ± 1 ms, respectively. Though ionization initially forms the cation radical, the radicals detected for (dA)20 are quantitatively identified as N6-deprotonated adenyl radicals by their absorption spectrum, which is computed quantum mechanically employing TD-DFT. Theoretical calculations show that deprotonation of the cation radical induces only weak spectral changes, in line with the spectra of the adenyl radical cation and the deprotonated radical trapped in low temperature glasses.

  2. Formation of fluorescent polydopamine dots from hydroxyl radical-induced degradation of polydopamine nanoparticles.

    Science.gov (United States)

    Lin, Jia-Hui; Yu, Cheng-Ju; Yang, Ya-Chun; Tseng, Wei-Lung

    2015-06-21

    This study describes the synthesis of fluorescent polydopamine dots (PDs) through hydroxyl radical-induced degradation of polydopamine nanoparticles. The decomposition of polydopamine nanoparticles to fluorescent PDs was confirmed using transmission electron microscopy and dark-field microscopy. The analysis of PDs by using laser desorption/ionization time-of-flight mass spectrometry revealed that the PDs consisted of dopamine, 5,6-dihydroxyindole, and trihydroxyindole units. Oligomerization and self-assembly of these units produced a broad adsorption band, resulting in an excitation-wavelength-dependent emission behavior. The maximal fluorescence of PDs appeared at 440 nm with a quantum yield of 1.2%. The coordination between the catechol groups of PDs and ferric ions (Fe(3+)) quenched the fluorescence of PDs; the limit of detection at a signal-to-noise ratio of 3 for Fe(3+) was determined to be 0.3 μM. The presence of pyrophosphate switched on the fluorescence of the PD-Fe(3+) complexes. Compared to the other reported methods for sensing Fe(3+), PDs provided simple, low-cost, and reusable detection of Fe(3+).

  3. Radical-induced chemistry from VUV photolysis of interstellar ice analogues containing formaldehyde

    Science.gov (United States)

    Butscher, Teddy; Duvernay, Fabrice; Danger, Grégoire; Chiavassa, Thierry

    2016-09-01

    Surface processes and radical chemistry within interstellar ices are increasingly suspected to play an important role in the formation of complex organic molecules (COMs) observed in several astrophysical regions and cometary environments. We present new laboratory experiments on the low-temperature solid state formation of complex organic molecules - glycolaldehyde, ethylene glycol, and polyoxymethylene - through radical-induced reactivity from VUV photolysis of formaldehyde in water-free and water-dominated ices. Radical reactivity and endogenous formation of COMs were monitored in situ via infrared spectroscopy in the solid state and post photolysis with temperature programmed desorption (TPD) using a quadripole mass spectrometer. We show the ability of free radicals to be stored when formed at low temperature in water-dominated ices, and to react with other radicals or on double bonds of unsaturated molecules when the temperature increases. It experimentally confirms the role of thermal diffusion in radical reactivity. We propose a new pathway for formaldehyde polymerisation induced by HCO radicals that might explain some observations made by the Ptolemy instrument on board the Rosetta lander Philae. In addition, our results seem to indicate that H-atom additions on H2CO proceed preferentially through CH2OH intermediate radicals rather than the CH3O radical.

  4. EFFECT OF HYDROALCOHOLIC EXTRACT OF TERMINALIA CATAPPA L. (COMBRETACEAE ON FREE RADICALS INDUCED IN RAT BRAIN

    Directory of Open Access Journals (Sweden)

    Calderón, Abhel

    2013-07-01

    Full Text Available The hydroalcoholic extract of Terminalia catappa leaves was tested in vivo to determine its antioxidant activity. The objective was to determine the antioxidant effect of the extract of T. catappa against free radicals induced in rat brain. We worked with two groups, the control consisting of positive control and negative control subsets which were administered carbon tetrachloride (CTC 1 ml·Kg-1 body weight and 2 mL of physiological saline solution (PSS, respectively. The experimental group consisted of two subgroups treated with the hydroalcoholic extract of T. catappa + CTC: one with 0.8 mg of extract T. catappa/kg bodyweight and the other with 1.5 mg T. catappa/kg body weight, for seven days. In all groups was determined the amount of free radicals in the brain by the technique of thiobarbituric acid reactive substances. From the positive control was obtained an average concentration of 26.68 μg malondialdehyde/g brain tissue, and in the negative control an average concentration of 9.76 μg malondialdehyde/g brain tissue; in the experimental group treated with T. catappa to 0.8 mg·kg-1 body weight and 1.5 mg·kg-1 body weight was obtained an average concentration of 13.64 μg malondialdehyde/g brain tissue and 15.80 μg malondialdehyde/g brain tissue, respectively. It was determined that the group treated with the hydroalcoholic extract of T. catappa concentration to 0.8 mg·Kg-1 body weight showed significant inhibition of free radicals compared to the positive control subgroup.

  5. Gene sensitizes cancer cells to chemotherapy drugs

    Science.gov (United States)

    NCI scientists have found that a gene, Schlafen-11 (SLFN11), sensitizes cells to substances known to cause irreparable damage to DNA.  As part of their study, the researchers used a repository of 60 cell types to identify predictors of cancer cell respons

  6. American Society of Gene & Cell Therapy

    Science.gov (United States)

    ... agencies, foundations, biotechnology and pharmaceutical companies. Mission: To advance knowledge, awareness, and education leading to the discovery and clinical application of gene and cell therapies to alleviate human disease. Vision: ASGCT will serve ...

  7. Microanalysis of gene expression in cultured cells

    NARCIS (Netherlands)

    E. van der Veer (Eveliene)

    1982-01-01

    textabstractIn this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing g

  8. Stem Cell-Based Gene Therapy.

    Science.gov (United States)

    Bagnis; Mannoni

    1997-01-01

    Many researchers and clinicians wonder if gene therapy remains a way to treat genetic or acquired life-threatening diseases. For the last few years, many experimental, pre-clinical, and clinical data have been published showing that it is possible to transfer with relatively high efficiency new genetic information (transgene) in many cells or tissues including both hematopoietic progenitor cells and differentiated cells. Based on experimental works, addition of the normal gene to cells with deletions, mutations, or alterations of the corresponding endogenous one has been shown to reverse the phenotype and to restore (in some case) the functional defect. In spite of very attractive preliminary results, however, suggesting the feasibility and safety of this process, therapeutically efficient gene transfer and expression in targeted cells or tissues must be proven. In this review, we will focus primarily on the attempts to use gene transfer in hematopoietic stem cells as a model for more general genetic manipulations of stem cells. Hematopoietic stem cells are included in a subset of bone marrow, cord blood, or peripheral blood cells identified by the expression of the CD34 antigen on their membrane.

  9. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  10. Hydroxyl-radical-induced oxidation of 5-methylcytosine in isolated and cellular DNA.

    Science.gov (United States)

    Madugundu, Guru S; Cadet, Jean; Wagner, J Richard

    2014-06-01

    The methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the regulation of genes during cell differentiation, embryogenesis and carcinogenesis. Despite its low abundance, 5-methylcytosine (5mC) is a hotspot for mutations in mammalian cells. Here, we measured five oxidation products of 5mC together with the analogous products of cytosine and thymine in DNA exposed to ionizing radiation in oxygenated aqueous solution. The products can be divided into those that arise from hydroxyl radical (•OH) addition at the 5,6-double bond of 5mC (glycol, hydantoin and imidazolidine products) and those that arise from H-atom abstraction from the methyl group of 5mC including 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC). Based on the analysis of these products, we show that the total damage at 5mC is about 2-fold greater than that at C in identical sequences. The formation of hydantoin products of 5mC is favored, compared to analogous reactions of thymine and cytosine, which favor the formation of glycol products. The distribution of oxidation products is sequence dependent in specific ODN duplexes. In the case of 5mC, the formation of 5hmC and 5fC represents about half of the total of •OH-induced oxidation products of 5mC. Several products of thymine, cytosine, 5mC, as well as 8-oxo-7,8-dihydroguanine (8oxoG), were also estimated in irradiated cells.

  11. Gene expression profiles in irradiated cancer cells

    Science.gov (United States)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-01

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  12. Gene expression profiles in irradiated cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C. [IBFM CNR - LATO, Cefalù, Segrate (Italy)

    2013-07-26

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  13. Genes involved in cell division in mycoplasmas

    Directory of Open Access Journals (Sweden)

    Frank Alarcón

    2007-01-01

    Full Text Available Bacterial cell division has been studied mainly in model systems such as Escherichia coli and Bacillus subtilis, where it is described as a complex process with the participation of a group of proteins which assemble into a multiprotein complex called the septal ring. Mycoplasmas are cell wall-less bacteria presenting a reduced genome. Thus, it was important to compare their genomes to analyze putative genes involved in cell division processes. The division and cell wall (dcw cluster, which in E. coli and B. subtilis is composed of 16 and 17 genes, respectively, is represented by only three to four genes in mycoplasmas. Even the most conserved protein, FtsZ, is not present in all mycoplasma genomes analyzed so far. A model for the FtsZ protein from Mycoplasma hyopneumoniae and Mycoplasma synoviae has been constructed. The conserved residues, essential for GTP/GDP binding, are present in FtsZ from both species. A strong conservation of hydrophobic amino acid patterns is observed, and is probably necessary for the structural stability of the protein when active. M. synoviae FtsZ presents an extended amino acid sequence at the C-terminal portion of the protein, which may participate in interactions with other still unknown proteins crucial for the cell division process.

  14. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  15. Optogenetics for gene expression in mammalian cells.

    Science.gov (United States)

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  16. OH-radical induced degradation of hydroxybenzoic- and hydroxycinnamic acids and formation of aromatic products-A gamma radiolysis study

    Energy Technology Data Exchange (ETDEWEB)

    Krimmel, Birgit; Swoboda, Friederike [University of Vienna, Department of Nutritional Sciences, Section Radiation Biology (Austria); Solar, Sonja, E-mail: sonja.solar@univie.ac.a [University of Vienna, Department of Nutritional Sciences, Section Radiation Biology (Austria); Reznicek, Gottfried [Department of Pharmacognosy, Althanstrasse 14, A-1090 Vienna (Austria)

    2010-12-15

    The OH-radical induced degradation of hydroxybenzoic acids (HBA), hydroxycinnamic acids (HCiA) and methoxylated derivatives, as well as of chlorogenic acid and rosmarinic acid was studied by gamma radiolysis in aerated aqueous solutions. Primary aromatic products resulting from an OH-radical attachment to the ring (hydroxylation), to the position occupied by the methoxyl group (replacement -OCH{sub 3} by -OH) as well as to the propenoic acid side chain of the cinnamic acids (benzaldehyde formations) were analysed by HPLC-UV and LC-ESI-MS. A comparison of the extent of these processes is given for 3,4-dihydroxybenzoic acid, vanillic acid, isovanillic acid, syringic acid, cinnamic acid, 4-hydroxycinnamic acid, caffeic acid, ferulic acid, isoferulic acid, chlorogenic acid, and rosmarinic acid. For all cinnamic acids and derivatives benzaldehydes were significant oxidation products. With the release of caffeic acid from chlorogenic acid the cleavage of a phenolic glycoside could be demonstrated. Reaction mechanisms are discussed.

  17. OH-radical induced degradation of hydroxybenzoic- and hydroxycinnamic acids and formation of aromatic products—A gamma radiolysis study

    Science.gov (United States)

    Krimmel, Birgit; Swoboda, Friederike; Solar, Sonja; Reznicek, Gottfried

    2010-12-01

    The OH-radical induced degradation of hydroxybenzoic acids (HBA), hydroxycinnamic acids (HCiA) and methoxylated derivatives, as well as of chlorogenic acid and rosmarinic acid was studied by gamma radiolysis in aerated aqueous solutions. Primary aromatic products resulting from an OH-radical attachment to the ring (hydroxylation), to the position occupied by the methoxyl group (replacement -OCH 3 by -OH) as well as to the propenoic acid side chain of the cinnamic acids (benzaldehyde formations) were analysed by HPLC-UV and LC-ESI-MS. A comparison of the extent of these processes is given for 3,4-dihydroxybenzoic acid, vanillic acid, isovanillic acid, syringic acid, cinnamic acid, 4-hydroxycinnamic acid, caffeic acid, ferulic acid, isoferulic acid, chlorogenic acid, and rosmarinic acid. For all cinnamic acids and derivatives benzaldehydes were significant oxidation products. With the release of caffeic acid from chlorogenic acid the cleavage of a phenolic glycoside could be demonstrated. Reaction mechanisms are discussed.

  18. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  19. Gene Therapy: a Breakthrough for Sickle Cell Anemia?

    Science.gov (United States)

    ... fullstory_163849.html Gene Therapy: A Breakthrough for Sickle Cell Anemia? But treatment has only been given to ... gene therapy to treat, or even potentially cure, sickle cell anemia. The findings come from just one patient, ...

  20. Ankylosing Spondylitis: From Cells to Genes

    Science.gov (United States)

    Zambrano-Zaragoza, José Francisco; Agraz-Cibrian, Juan Manuel; González-Reyes, Christian; Durán-Avelar, Ma. de Jesús; Vibanco-Pérez, Norberto

    2013-01-01

    Ankylosing spondylitis (AS) is a chronic inflammatory disease of unknown etiology, though it is considered an autoimmune disease. HLA-B27 is the risk factor most often associated with AS, and although the mechanism of involvement is unclear, the subtypes and other features of the relationship between HLA-B27 and AS have been studied for years. Additionally, the key role of IL-17 and Th17 cells in autoimmunity and inflammation suggests that the latter and the cytokines involved in their generation could play a role in the pathogenesis of this disease. Recent studies have described the sources of IL-17 and IL-23, as well as the characterization of Th17 cells in autoimmune diseases. Other cells, such as NK and regulatory T cells, have been implicated in autoimmunity and have been evaluated to ascertain their possible role in AS. Moreover, several polymorphisms, mutations and deletions in the regulatory proteins, protein-coding regions, and promoter regions of different genes involved in immune responses have been discovered and evaluated for possible genetic linkages to AS. In this review, we analyze the features of HLA-B27 and the suggested mechanisms of its involvement in AS while also focusing on the characterization of the immune response and the identification of genes associated with AS. PMID:23970995

  1. Silencing of Bcl-2 gene expression by siRNA transfection inhibits the protective effect of fluvastatin against cell apoptosis in human aortic endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Wenwen Zhong; Yang Liu; Jian Li; Hui Tian

    2008-01-01

    Objective To study the protective effect of fluvastatin,one of the HMG-CoA reductase inhibitors (statins),against oxygen radical-induced oxidative damages in human aortic endothelial cell,and the role of Bcl-2 in this protection.Methods Human aortic endothelial cells with or without Bcl-2 siRNA transfection were subjected to 1-100 nM of fluvastatin and 100 la hydrogen peroxide for 24 hours.Bcl-2 mRNA and protein expression were measured by Taqman quantitative PCR and Western blotting.Cell apoptosis was measured by normal and fluorescent microscopy and Cell Death Detection ELISA.Results In the Bcl-2-expressed cells,fluvastatin significantly reversed hydrogen peroxide-induced microscopic apoptosis and apoptotic DNA fragmentation,which were accompanied by a markedly upregulation of Bcl-2 expression by fluvastatin.However,the endothelial protection by fluvastatin was completely lost in Bcl-2 siRNA transfected cells.Conclusion Fluvastatin protects human endothelial cells against oxygen radical-induced cell apoptosis in vitro,and this protection seemed to be mediated in a Bcl-2 dependent pathway.(J Geriatr Cardil 12008;5:33-38)

  2. Synthesis of carba sugars from aldonolactones. Part IV. Stereospecific synthesis of carbaheptopyranoses by radical-induced carbocyclisation of 2,3-unsaturated octonolactones

    DEFF Research Database (Denmark)

    Wagner, Sussi Holstein; Lundt, Inge

    2001-01-01

    -manno-configuration, respectively. The key step was a regio- and stereoselective 6-exo-trig radical-induced carbocyclisation of the unsaturated octonolactones to give bicyclic cyclohexane-lactone derivatives. Reduction of the lactone moiety using Ca(BH4)(2) gave the said carbaheptopyranoses. The 8-bromo-8-deoxy-2,3-unsaturated...

  3. Titanocene(III) chloride mediated radical induced addition-elimination route to the synthesis of racemic and optically active trisubstituted tetrahydrofurans: Formal synthesis of magnofargesin and 7'-epimagnofargesin

    Indian Academy of Sciences (India)

    P CHAKRABORTY; S K MANDAL; S C ROY

    2016-07-01

    Titanocene(III) Chloride mediated radical induced synthesis of 4-benzylidene substituted tetrahydrofuran, a typical lignan skeleton, has been accomplished in good yield through addition-elimination route in racemic as well as in optically active forms. The method has been applied to the synthesis of furano lignans, magnofargesin (1) and 7'-epimagnofargesin (2) in optically active forms.

  4. GM-CSF GENE OR B7-1 GENE MODIFIED MURINE EL-4 CELLS VACCINE

    Institute of Scientific and Technical Information of China (English)

    张清媛; 李殿俊; 王志华

    2001-01-01

    Objective: To study the vaccine potency of gene-modified tumor cells. Methods: The EL-4 lymphoma was transduced with recombinant retrovirus containing the murine GM-CSF gene or B7-1 gene. The effect of gene transduction on antitumor immunity was investigated. Results: Flow cytometry analysis showed that expression of their surface marker between wild-type EL-4 cells and gene transduced tumor cells was the same except for CD80 positive in B7-1 gene transduced cells. GM-CSF gene or B7-1 gene transduced EL-4 cells resulted in remarkable loss of tumorigenicity in syngenetic mice. The systemic protective immunity was induced against the challenge with EL-4/wt cells. Therapeutic vaccine with EL-4/GM-CSF or EL/7-1 cells could retard the growth of established early-stage EL-4/wt tumor significantly, but not retard the growth of late-stage EL-4/wt tumor. Irradiated GM-CSF gene transduced EL-4 cells showed strong vaccine effect against EL-4 cell challenge, but irradiated B7-1 gene transduced EL-4 cells showed weak vaccine effect. Remarkable cooperative antitumor effect against EL-4 cell challenge was observed when both irradiated EL-4/GM-CSF and EL-4/B7-1 were inoculated together. Conclusion: GM-CSF gene or B7-1 gene transduced combination of the two kinds of vaccine may have potential application value in human cancer treatment.

  5. Gene-modified bone marrow cell therapy for prostate cancer.

    Science.gov (United States)

    Wang, H; Thompson, T C

    2008-05-01

    There is a critical need to develop new and effective cancer therapies that target bone, the primary metastatic site for prostate cancer and other malignancies. Among the various therapeutic approaches being considered for this application, gene-modified cell-based therapies may have specific advantages. Gene-modified cell therapy uses gene transfer and cell-based technologies in a complementary fashion to chaperone appropriate gene expression cassettes to active sites of tumor growth. In this paper, we briefly review potential cell vehicles for this approach and discuss relevant gene therapy strategies for prostate cancer. We further discuss selected studies that led to the conceptual development and preclinical testing of IL-12 gene-modified bone marrow cell therapy for prostate cancer. Finally, we discuss future directions in the development of gene-modified cell therapy for metastatic prostate cancer, including the need to identify and test novel therapeutic genes such as GLIPR1.

  6. Direct cell lysis for single-cell gene expression profiling

    Directory of Open Access Journals (Sweden)

    David eSvec

    2013-11-01

    Full Text Available The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA to be the best lysis agent, resulting in efficient cell lysis, high RNA stability and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single cells as well as samples composed of small numbers of cells.

  7. Gene therapy of primary T cell immunodeficiencies.

    Science.gov (United States)

    Fischer, Alain; Hacein-Bey-Abina, Salima; Cavazzana-Calvo, Marina

    2013-08-10

    Gene therapy of severe combined immunodeficiencies has been proven to be effective to provide sustained correction of the T cell immunodeficiencies. This has been achieved for 2 forms of SCID, i.e SCID-X1 (γc deficiency) and adenosine deaminase deficiency. Occurrence of gene toxicity generated by integration of first generation retroviral vectors, as observed in the SCID-X1 trials has led to replace these vectors by self inactivated (SIN) retro(or lenti) viruses that may provide equivalent efficacy with a better safety profile. Results of ongoing clinical studies in SCID as well as in other primary immunodeficiencies, such as the Wiskott Aldrich syndrome, will be thus very informative.

  8. Hydroxyl radicals induced by quartz particles in lung alveolar macrophages: the role of surface iron

    Institute of Scientific and Technical Information of China (English)

    LI Yi; ZHU Tong; GUO Xinbiao; SHANG Yu

    2006-01-01

    Previous studies have shown that hydroxyl radical generation is a key step in the mechanism of pathogenic process caused by airborne particles to the lung. However, there is no direct evidence for dose-response relationship between airborne particles and hydroxyl radical generation. In this study, hydroxyl radicals generated in lung alveolar macrophages exposed to quartz particles were measured using a highly sensitive capillary electrophoresis-fluorescence detection method. The results demonstrated that quartz particles induced the generation of hydroxyl radical in a dose-dependent manner, and the amount of the hydroxyl radicals was 10-10 mol/106 cells.The viability of alveolar macrophages exposed to quartz particles decreased with the increase of quartz concentration, showing a clear doseresponse relationship. Hydroxyl radical scavenger mannitol could increase the viability of quartz-treated cells, suggesting that hydroxyl radical contributed directly to cell death. In this study this contribution accounted for about 5%-20% of cell death. The hydroxyl radical generating potential was found to be related to surface iron content of the quartz particles.

  9. Rheumatoid factors, B cells and immunoglobulin genes.

    Science.gov (United States)

    Jefferis, R

    1995-04-01

    The paradigm of self, non-self discrimination in the immune system is under review as autoreactive B or T cells are increasingly delineated within normal individuals. The products of autoreactive B cells are, mostly, polyspecific IgM antibodies of low affinity. These 'natural' antibodies include rheumatoid factors (RF) encoded by unmutated germline immunoglobulin genes. In rheumatoid arthritis (RA) the RF may be of the IgM, IgG or IgA isotype, show evidence of somatic mutation and have increased affinity; consistent with maturation of an antigen driven immune response. This response could be initiated or driven by an auto-immunogenic form of IgG or an exogenous cross-reactive antigen. Changes in galactosylation of IgG have been reported to be a valuable diagnostic and prognostic indicator in RA. Speculation that these changes may precipitate some of the disease processes is critically reviewed.

  10. Global Identification of Disease Associated Genes in Fragile X Cells

    Science.gov (United States)

    2016-08-01

    AWARD NUMBER: W81XWH-15-1-0204 TITLE: Global Identification of Disease-Associated Genes in Fragile X Cells PRINCIPAL INVESTIGATOR: Wenyi Feng...Global Identification of Disease-Associated Genes in Fragile X Cells 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-15-1-0204 GRANT1171 2389... genes in fragile X cells compared to normal cells. o What was accomplished under these goals? Below I list the experiments and conclusions for each goal

  11. THE GENE EXPRESSION PROFILE OF HIGHLY METASTATIC HUMAN OVARIAN CANCER CELL LINE BY GENE CHIP

    Institute of Scientific and Technical Information of China (English)

    吕桂泉; 许沈华; 牟瀚舟; 朱赤红; 羊正炎; 高永良; 楼洪坤; 刘祥麟; 杨文; 程勇

    2001-01-01

    To study the gene expression of high metastatic human ovarian carcinoma cell line (HO-8910PM) and to screen for novel metastasis- associated genes by cDNA microarray. Methods: The cDNA was retro-transcribed from equal quantity mRNA derived from tissues of highly metastatic ovarian carcinoma cell line and normal ovarian, and was labeled with Cy5 and Cy3 fluorescence as probes. The mixed probes were hybridized with BioDoor 4096 double dot human whole gene chip. The chip was scanned by scanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. Results: By applying the cDNA microarray we found: A total of 323 genes whose expression level were 3 times higher or lower in HO-8910PM cell than normal ovarian epithelium cell were screened out, with 71 higher and 252 lower respectively. Among these 10 were new genes. 67 genes showed expression difference bigger than 6 times between HO-8910PM cell and normal ovarian epithelium cell, among these genes 12 were higher, 55 lower, and two new genes were found. Conclusion: cDNA microarray technique is effective in screening the differentially expressed genes between human ovarian cancer cell line (HO-8910PM) and normal ovarian epithelium cell. Using the cDNA microarray to analyze of human ovarian cancer cell line gene expression profile difference will help the gene diagnosis, treatment and protection.

  12. Cell cycle-dependent gene networks relevant to cancer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The analysis of sophisticated interplays between cell cycle-dependent genes in a disease condition is one of the largely unexplored areas in modern tumor biology research. Many cell cycle-dependent genes are either oncogenes or suppressor genes, or are closely asso- ciated with the transition of a cell cycle. However, it is unclear how the complicated relationships between these cell cycle-dependent genes are, especially in cancers. Here, we sought to identify significant expression relationships between cell cycle-dependent genes by analyzing a HeLa microarray dataset using a local alignment algorithm and constructed a gene transcriptional network specific to the cancer by assembling these newly identified gene-gene relationships. We further characterized this global network by partitioning the whole network into several cell cycle phase-specific sub-networks. All generated networks exhibited the power-law node-degree dis- tribution, and the average clustering coefficients of these networks were remarkably higher than those of pure scale-free networks, indi- cating a property of hierarchical modularity. Based on the known protein-protein interactions and Gene Ontology annotation data, the proteins encoded by cell cycle-dependent interacting genes tended to share the same biological functions or to be involved in the same biological processes, rather than interacting by physical means. Finally, we identified the hub genes related to cancer based on the topo- logical importance that maintain the basic structure of cell cycle-dependent gene networks.

  13. Raman tweezers spectroscopy study of free radical induced oxidative stress leading to eryptosis

    Science.gov (United States)

    Barkur, Surekha; Bankapur, Aseefhali; Chidangil, Santhosh

    2016-11-01

    Raman tweezers spectroscopy study of effect of free radicals was carried out on erythrocytes. We prepared hydroxyl radicals using Fenton reaction (which yields hydroxyl radicals). Raman spectra were acquired from single, trapped erythrocytes after supplementing with these free radicals. The changes in the Raman bands such as 1211 cm-1, 1224 cm-1, 1375 cm-1 indicate deoxygenation of red blood cells (RBCs). Our study shows that free radicals can induce oxidative stress on erythrocytes. The changes in the Raman spectra as well as shape of erythrocytes indicate that oxidative stress can trigger eryptosis in erythrocytes.

  14. Msh homeobox genes regulate cadherin-mediated cell adhesion and cell-cell sorting.

    Science.gov (United States)

    Lincecum, J M; Fannon, A; Song, K; Wang, Y; Sassoon, D A

    1998-07-01

    Msx-1 and Msx-2 are two closely related homeobox genes expressed in cephalic neural crest tooth buds, the optic cup endocardial cushions, and the developing limb [Hill and Davidson, 1991; Monaghan et al., 1991; Robert et al., 1991]. These sites correspond to regions of active cell segregation and proliferation under the influence of epithelial-mesenchymal cell interactions [Brown et al., 1993; Davidson et al., 1991], suggesting that Msx-1 and Msx-2 regulate cell-cell interactions. We have investigated the potential relationship between expression of the Msh homeobox genes (Msx-1 and Msx-2) and cadherin-mediated cell adhesion and cell sorting. We report that cell lines stably expressing Msx-1 or Msx-2 differentially sort on the basis of Msh gene expression. We demonstrate in vitro that initial cell aggregation involves calcium-dependent adhesion molecules (cadherins) and that Msh genes regulate cadherin-mediated adhesion. These results support the hypothesis that Msh genes play a role in the regulation of cell-cell adhesion and provide a link between the genetic phenomena of homeobox gene expression and cellular events involved in morphogenesis, including cell sorting and proliferation.

  15. Toward stable gene expression in CHO cells

    Science.gov (United States)

    Mariati; Koh, Esther YC; Yeo, Jessna HM; Ho, Steven CL; Yang, Yuansheng

    2014-01-01

    Maintaining high gene expression level during long-term culture is critical when producing therapeutic recombinant proteins using mammalian cells. Transcriptional silencing of promoters, most likely due to epigenetic events such as DNA methylation and histone modifications, is one of the major mechanisms causing production instability. Previous studies demonstrated that the core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene is effective to prevent DNA methylation. We generated one set of modified human cytomegalovirus (hCMV) promoters by insertion of one or two copies of IE in either forward or reverse orientations into different locations of the hCMV promoter. The modified hCMV with one copy of IE inserted between the hCMV enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability in CHO cells without comprising expression level when compared with the wild type hCMV. We also found that insertion of IE into a chimeric murine CMV (mCMV) enhancer and human elongation factor-1α core (hEF) promoter in reverse orientation did not enhance expression stability, indicating that the effect of IE on expression stability is possibly promoter specific. PMID:25482237

  16. Hydroxyl-radical-induced degradative oxidation of beta-lactam antibiotics in water: absolute rate constant measurements.

    Science.gov (United States)

    Dail, Michelle K; Mezyk, Stephen P

    2010-08-19

    The beta-lactam antibiotics are some of the most prevalent pharmaceutical contaminants currently being detected in aquatic environments. Because the presence of any trace level of antibiotic in water may adversely affect aquatic ecosystems and contribute to the production of antibiotic-resistant bacteria, active removal by additional water treatments, such as using advanced oxidation and reduction processes (AO/RPs), may be required. However, to ensure that any AOP treatment process occurs efficiently and quantitatively, a full understanding of the kinetics and mechanisms of all of the chemical reactions involved under the conditions of use is necessary. In this study, we report on our kinetic measurements for the hydroxyl-radical-induced oxidation of 11 beta-lactam antibiotics obtained using electron pulse radiolysis techniques. For the 5-member ring species, an average reaction rate constant of (7.9 +/- 0.8) x 10(9) M(-1) s(-1) was obtained, slightly faster than for the analogous 6-member ring containing antibiotics, (6.6 +/- 1.2) x 10(9) M(-1) s(-1). The consistency of these rate constants for each group infers a common reaction mechanism, consisting of the partitioning of the hydroxyl radical between addition to peripheral aromatic rings and reaction with the central double-ring core of these antibiotics.

  17. HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFER TO LEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    FU Jian-xin; CHEN Zi-xing; CEN Jian-nong; WANG Wei; RUAN Chang-geng

    1999-01-01

    Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1.The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR).Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days' culture.Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

  18. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line

    Institute of Scientific and Technical Information of China (English)

    ZOU Hong-yun; MA Li; MENG Min-jie; YAO Xin-sheng; LIN Ying; WU Zhen-qiang; HE Xiao-wei; WANG Ju-fang; WANG Xiao-ning

    2007-01-01

    Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor (TCR) gene recombination.Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVβ chain was examined by the TCR GeneScan technique.Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ25' and Dβ 23' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation.Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire. However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  19. Protective effects of boldine against free radical-induced erythrocyte lysis.

    Science.gov (United States)

    Jiménez, I; Garrido, A; Bannach, R; Gotteland, M; Speisky, H

    2000-08-01

    Boldine, an aporphine alkaloid extracted from the leaves and bark of boldo (Peumus boldus Mol.), has been shown to exhibit strong free-radical scavenger and antioxidant properties. Here, we report the in vitro ability of boldine to protect intact red cells against the haemolytic damage induced by the free radical initiator 2, 2'-azobis-(2-amidinopropane) (AAPH). Boldine concentration-dependently prevented the AAPH-induced leakage of haemoglobin into the extracellular medium. Substantial and similar cyto-protective effects of boldine were observed whether the antioxidant was added 1 h prior to, or simultaneously with, the azo-compound. The delayed addition of boldine, by 1 h relative to AAPH, diminished but did not abolish its cytoprotective effect. However, negligible effects of boldine were observed after its addition to erythrocytes previously incubated with AAPH for 2 h. The data presented demonstrate that, in addition to its well-established antioxidant effects, boldine also displays time-dependently strong cytoprotective properties against chemically induced haemolytic damage. Copyright 2000 John Wiley & Sons, Ltd.

  20. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line.

    Science.gov (United States)

    Zou, Hong-yun; Ma, Li; Meng, Min-jie; Yao, Xin-sheng; Lin, Ying; Wu, Zhen-qiang; He, Xiao-wei; Wang, Ju-fang; Wang, Xiao-ning

    2007-03-05

    Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether thebreceptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkathuman T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of Tcell receptor (TCR) gene recombination. TCR Dbeta-Jbeta signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVbeta chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVbeta chain was examined by the TCR GeneScan technique. RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dbeta2-Jbeta2 signal joints and ds RSS breaks associated with the Dbeta2 5' and Dbeta 2 3' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVbeta chain did not change during cell proliferation. RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  1. Alterations of FHIT Gene and P16 Gene in Nickel Transformed Human Bronchial Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    WEI-DONG JI; JIA-KUN CHEN; JIA-CHUN LU; ZHONG-LIANG WU; FEI YI; SU-MEI FENG

    2006-01-01

    To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immoral human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis. Methods 16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting. Results NiS-treated cells exhibited overlapping growth. Compared with that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P<0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in p16 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8. Conclusions The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

  2. Regulated genes in mesenchymal stem cells and gastriccancer

    Institute of Scientific and Technical Information of China (English)

    Shihori Tanabe; Kazuhiko Aoyagi; Hiroshi Yokozaki; Hiroki Sasaki

    2015-01-01

    AIM To investigate the genes regulated in mesenchymalstem cells (MSCs) and diffuse-type gastric cancer (GC),gene expression was analyzed.METHODS: Gene expression of MSCs and diffuse-typeGC cells were analyzed by microarray. Genes relatedto stem cells, cancer and the epithelial-mesenchymaltransition (EMT) were extracted from human genelists using Gene Ontology and reference information.Gene panels were generated, and messenger RNAgene expression in MSCs and diffuse-type GC cells wasanalyzed. Cluster analysis was performed using the NCSSsoftware.RESULTS: The gene expression of regulator of G-proteinsignaling 1 (RGS1) was up-regulated in diffuse-type GCcells compared with MSCs. A panel of stem-cell relatedgenes and genes involved in cancer or the EMT wereexamined. Stem-cell related genes, such as growtharrest-specific 6, musashi RNA-binding protein 2 andhairy and enhancer of split 1 (Drosophila), NOTCHfamily genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated,and genes related to stem cells and NOTCH signalingare altered in diffuse-type GC compared with MSCs.

  3. Gene expression profile of renal cell carcinoma clear cell type

    Directory of Open Access Journals (Sweden)

    Marcos F. Dall’Oglio

    2010-08-01

    Full Text Available PURPOSE: The determination of prognosis in patients with renal cell carcinoma (RCC is based, classically, on stage and histopathological aspects. The metastatic disease develops in one third of patients after surgery, even in localized tumors. There are few options for treating those patients, and even the new target designed drugs have shown low rates of success in controlling disease progression. Few studies used high throughput genomic analysis in renal cell carcinoma for determination of prognosis. This study is focused on the identification of gene expression signatures in tissues of low-risk, high-risk and metastatic RCC clear cell type (RCC-CCT. MATERIALS AND METHODS: We analyzed the expression of approximately 55,000 distinct transcripts using the Whole Genome microarray platform hybridized with RNA extracted from 19 patients submitted to surgery to treat RCC-CCT with different clinical outcomes. They were divided into three groups (1 low risk, characterized by pT1, Fuhrman grade 1 or 2, no microvascular invasion RCC; (2 high risk, pT2-3, Fuhrman grade 3 or 4 with, necrosis and microvascular invasion present and (3 metastatic RCC-CCT. Normal renal tissue was used as control. RESULTS: After comparison of differentially expressed genes among low-risk, high-risk and metastatic groups, we identified a group of common genes characterizing metastatic disease. Among them Interleukin-8 and Heat shock protein 70 were over-expressed in metastasis and validated by real-time polymerase chain reaction. CONCLUSION: These findings can be used as a starting point to generate molecular markers of RCC-CCT as well as a target for the development of innovative therapies.

  4. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control.

    Science.gov (United States)

    Grant, Gavin D; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K; Mahoney, J Matthew; Loros, Jennifer J; Dunlap, Jay C; Whitfield, Michael L

    2012-08-01

    We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

  5. Random matrix analysis for gene interaction networks in cancer cells

    CERN Document Server

    Kikkawa, Ayumi

    2016-01-01

    Motivation: The investigation of topological modifications of the gene interaction networks in cancer cells is essential for understanding the desease. We study gene interaction networks in various human cancer cells with the random matrix theory. This study is based on the Cancer Network Galaxy (TCNG) database which is the repository of huge gene interactions inferred by Bayesian network algorithms from 256 microarray experimental data downloaded from NCBI GEO. The original GEO data are provided by the high-throughput microarray expression experiments on various human cancer cells. We apply the random matrix theory to the computationally inferred gene interaction networks in TCNG in order to detect the universality in the topology of the gene interaction networks in cancer cells. Results: We found the universal behavior in almost one half of the 256 gene interaction networks in TCNG. The distribution of nearest neighbor level spacing of the gene interaction matrix becomes the Wigner distribution when the net...

  6. DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions.

    OpenAIRE

    Satoh, M S; C. J. Jones; Wood, R D; Lindahl, T

    1993-01-01

    Plasmid DNA was gamma-irradiated or treated with H2O2 in the presence of Cu2+ to generate oxygen free radical-induced lesions. Open circular DNA molecules were removed by ethidium bromide/CsCl density gradient centrifugation. The closed circular DNA fraction was treated with the Escherichia coli reagent enzymes endonuclease III (Nth protein) and Fpg protein. This treatment converted DNA molecules containing the major base lesions pyrimidine hydrates and 8-hydroxyguanine to a nicked form. Rema...

  7. Progress in gene transfer by germ cells in mammals

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer (MGCMGT)or female germ cell mediated gene transfer(FGCMGT)technique.Sperm-mediated gene transfer (SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been extensively used for producing transgenic animals.The newly developed approach of FGCMGT,ovary-mediated gene transfer(OMGT) is also a novel and useful tool for efficient transgenesis.This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques,methods developed and mechanisms of nucleic acid uptake by germ cells.

  8. Gene pair signatures in cell type transcriptomes reveal lineage control

    Science.gov (United States)

    Heinäniemi, Merja; Nykter, Matti; Kramer, Roger; Wienecke-Baldacchino, Anke; Sinkkonen, Lasse; Zhou, Joseph Xu; Kreisberg, Richard; Kauffman, Stuart A.; Huang, Sui; Shmulevich, Ilya

    2013-01-01

    The distinct cell types of multicellular organisms arise due to constraints imposed by gene regulatory networks on the collective change of gene expression across the genome, creating self-stabilizing expression states, or attractors. We compiled a resource of curated human expression data comprising 166 cell types and 2,602 transcription regulating genes and developed a data driven method built around the concept of expression reversal defined at the level of gene pairs, such as those participating in toggle switch circuits. This approach allows us to organize the cell types into their ontogenetic lineage-relationships and to reflect regulatory relationships among genes that explain their ability to function as determinants of cell fate. We show that this method identifies genes belonging to regulatory circuits that control neuronal fate, pluripotency and blood cell differentiation, thus offering a novel large-scale perspective on lineage specification. PMID:23603899

  9. Isolating gene-corrected stem cells without drug selection.

    Science.gov (United States)

    Hatada, Seigo; Arnold, Larry W; Hatada, Tomoko; Cowhig, John E; Ciavatta, Dominic; Smithies, Oliver

    2005-11-08

    Progress in isolating stem cells from tissues, or generating them from adult cells by nuclear transfer, encourages attempts to use stem cells from affected individuals for gene correction and autologous therapy. Current viral vectors are efficient at introducing transgenic sequences but result in random integrations. Gene targeting, in contrast, can directly correct an affected gene, or incorporate corrective sequences into a site free from undesirable side effects, but efficiency is low. Most current targeting procedures, consequently, use positive-negative selection with drugs, often requiring >/=10 days. This drug selection causes problems with stem cells that differentiate in this time or require feeder cells, because the feeders must be drug resistant and so are not eliminated by the selection. To overcome these problems, we have developed a procedure for isolating gene-corrected stem cells free from feeder cells after 3-5 days culture without drugs. The method is still positive-negative, but the positive and negative drug-resistance genes are replaced with differently colored fluorescence genes. Gene-corrected cells are isolated by FACS. We tested the method with mouse ES cells having a mutant hypoxanthine phosphoribosyltransferase (Hprt) gene and grown on feeder cells. After 5 days in culture, gene-corrected cells were obtained free from feeder cells at a "purity" of >30%, enriched >2,000-fold and with a recovery of approximately 20%. Corrected cells were also isolated singly for clonal expansion. Our FACS-based procedure should be applicable at small or large scale to stem cells that can be cultured (with feeder cells, if necessary) for >/=3 days.

  10. Tracking gene-modified T cells in vivo.

    Science.gov (United States)

    Recchia, Alessandra; Mavilio, Fulvio

    2009-01-01

    Identification, monitoring, and analysis of genetically modified cells in the peripheral blood are an important component of the clinical follow-up of patients treated by hematopoietic cell gene therapy. Analysis of gene-marked peripheral blood cells provides crucial information on gene transfer efficiency as well as on the nature and characteristics of the genetically modified cells, and may provide early evidence of the occurrence of potentially detrimental side effects. T lymphocytes are a convenient target for this type of analysis, due to their abundance and their relatively long life span in vivo. Tracking of gene-marked T cells is based on relatively simple, FACS- and PCR-based techniques, which may be applied to monitoring genetically modified T cells as well as T cells derived from transplanted, genetically modified hematopoietic stem cells. This chapter provides a description of these techniques and clues to their rational use in a clinical setting.

  11. Removal of precursors for disinfection by-products (Dbps)--differences between ozone- and OH-radical-induced oxidation.

    Science.gov (United States)

    Kleiser, G; Frimmel, F H

    2000-06-22

    Pre-oxidation is often applied to reduce the formation of disinfection by-products (DBPs). The aim of pre-oxidation is to remove the centers of natural organic matter (NOM) which are responsible for the formation of DBPs. In this paper, the differences between ozone- and OH-radical-induced oxidation to remove DBP-precursors are compared. The experiments were done with water of the River Ruhr (Germany) with a concentration of dissolved organic carbon (DOC) of 2 mg/l. Ozonation was able to remove DBP precursors selectively. After application of an absorbed ozone mass of 1.5 mg/mg DOC, a reduction in the formation potential for (THM-FP) and in the formation potential for organic halogen adsorbable on activated carbon (AOX-FP) down to 68 and 73% of the initial concentration was achieved, respectively. A removal of NOM was not achieved using absorbed ozone masses between 0.5 and 1.5 mg/mg DOC. In the hydrogen peroxide/UV process, in which OH-radicals are the reactive species, an increase in the THM concentration was measured after application of this process with short irradiation times. The maximum value of the THM-FP was 20% higher than the initial THM-FP. After an irradiation time of 1,050 min and a hydrogen peroxide consumption of 5.6 mg/l, the THM-FP and AOX-FP decreased to 75 and 71% of the initial formation potential, respectively. There was no selective removal of DBP precursors because the DOC concentration decreased also to 75% of the initial DOC-concentration after 1,050 min of irradiation.

  12. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    Institute of Scientific and Technical Information of China (English)

    Yoshiyuki Takahara; Mitsuo Takahashi; Hiroki Wagatsuma; Fumihiko Yokoya; Qing-Wei Zhang; Mutsuyo Yamaguchi; Hiroyuki Aburatani; Norifumi Kawada

    2006-01-01

    AIM: To determine the gene expression profile data for the whole liver during development of dimethylnitrosamine (DMN)-induced hepatic fibrosis.METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells),and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells.RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSCspecific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis,suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocytespecific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis.CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis.

  13. Freedom of expression: cell-type-specific gene profiling.

    Science.gov (United States)

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  14. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    Science.gov (United States)

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  15. Microarray gene expression profiling and analysis in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sadhukhan Provash

    2004-06-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  16. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    Energy Technology Data Exchange (ETDEWEB)

    Cowan, Robert W. [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Ghert, Michelle [Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Department of Surgery, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Singh, Gurmit, E-mail: gurmit.singh@jcc.hhsc.ca [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These

  17. Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library

    Directory of Open Access Journals (Sweden)

    Eddy Edward M

    2007-07-01

    Full Text Available Abstract Background The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. Results We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. Conclusion We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

  18. Suicide genes: monitoring cells in patients with a safety switch

    OpenAIRE

    Eissenberg, Linda G.; Rettig, Michael; Dehdashti, Farrokh; Piwnica-Worms, David; John F. DiPersio

    2014-01-01

    Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical tr...

  19. Suicide genes: monitoring cells in patients with a safety switch

    OpenAIRE

    Linda Groppe Eissenberg; Michael eRettig; Farrokh eDehdashti; David ePiwnica-Worms; John F. DiPersio

    2014-01-01

    Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical t...

  20. On-Chip Integration of Cell-Free Gene Expression

    Science.gov (United States)

    Buxboim, Amnon; Morpurgo, Margherita; Bar-Dagan, Maya; Frydman, Veronica; Zbaida, David; Bar-Ziv, Roy

    2006-03-01

    We present a synthetic approach for the study of gene networks in vitro which is complementary to traditional in vivo methodologies. We have developed a technology for submicron integration of functional genes and on-chip protein synthesis using a cell-free transcription/translation system. The interaction between genes is facilitated by diffusion of on-chip gene expression products from `source' genes towards `acceptor' genes. Our technology is simple and inexpensive and can serve as an improved platform for a wide variety of protein and DNA biochip applications.

  1. Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yoshiaki Tabuchi

    2014-05-01

    Full Text Available Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF, we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I and Atf4 and Hspa5 (for Up-II. Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells.

  2. Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells

    Institute of Scientific and Technical Information of China (English)

    胡庆柳; 朴英杰; 邹飞

    2003-01-01

    Objective To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.Methods Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with α-32P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by AtlasImage analysis software Version 1.01a.Results Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells.Conclusion The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

  3. Runx Family Genes in Tissue Stem Cell Dynamics.

    Science.gov (United States)

    Wang, Chelsia Qiuxia; Mok, Michelle Meng Huang; Yokomizo, Tomomasa; Tergaonkar, Vinay; Osato, Motomi

    2017-01-01

    The Runx family genes play important roles in development and cancer, largely via their regulation of tissue stem cell behavior. Their involvement in two organs, blood and skin, is well documented. This review summarizes currently known Runx functions in the stem cells of these tissues. The fundamental core mechanism(s) mediated by Runx proteins has been sought; however, it appears that there does not exist one single common machinery that governs both tissue stem cells. Instead, Runx family genes employ multiple spatiotemporal mechanisms in regulating individual tissue stem cell populations. Such specific Runx requirements have been unveiled by a series of cell type-, developmental stage- or age-specific gene targeting studies in mice. Observations from these experiments revealed that the regulation of stem cells by Runx family genes turned out to be far more complex than previously thought. For instance, although it has been reported that Runx1 is required for the endothelial-to-hematopoietic cell transition (EHT) but not thereafter, recent studies clearly demonstrated that Runx1 is also needed during the period subsequent to EHT, namely at perinatal stage. In addition, Runx1 ablation in the embryonic skin mesenchyme eventually leads to complete loss of hair follicle stem cells (HFSCs) in the adult epithelium, suggesting that Runx1 facilitates the specification of skin epithelial stem cells in a cell extrinsic manner. Further in-depth investigation into how Runx family genes are involved in stem cell regulation is warranted.

  4. Genes affecting β-cell function in type 1 diabetes

    DEFF Research Database (Denmark)

    Fløyel, Tina; Kaur, Simranjeet; Pociot, Flemming

    2015-01-01

    Type 1 diabetes (T1D) is a multifactorial disease resulting from an immune-mediated destruction of the insulin-producing pancreatic β cells. Several environmental and genetic risk factors predispose to the disease. Genome-wide association studies (GWAS) have identified around 50 genetic regions...... that affect the risk of developing T1D, but the disease-causing variants and genes are still largely unknown. In this review, we discuss the current status of T1D susceptibility loci and candidate genes with focus on the β cell. At least 40 % of the genes in the T1D susceptibility loci are expressed in human...... islets and β cells, where they according to recent studies modulate the β-cell response to the immune system. As most of the risk variants map to noncoding regions of the genome, i.e., promoters, enhancers, intergenic regions, and noncoding genes, their possible involvement in T1D pathogenesis as gene...

  5. Suitability of endogenous reference genes for gene expression studies with human intraocular endothelial cells

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    Wei Ruoxin

    2013-02-01

    Full Text Available Abstract Background The use of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR has become widely applied as a method to measure transcript abundance. In order to be reflective of biological processes during health and disease this method is dependent on normalisation of data against stable endogenous controls. However, these genes can vary in their stability in different cell types. The importance of reference gene validation for a particular cell type is now well recognised and is an important step in any gene expression study. Results Cultured primary human choroidal and retinal endothelial cells were treated with the immunostimulant polyinosinic: polycytidylic acid or untreated. qRT-PCR was used to quantify the expression levels of 10 commonly used endogenous control genes, TBP, HPRT1, GAPDH, GUSB, PPIA, RPLP0, B2M, 18S rRNA, PGK1 and ACTB. Three different mathematical algorithms, GeNorm, NormFinder, and BestKeeper were used to analyse gene stability to give the most representative validation. In choroidal endothelial cells the most stable genes were ranked as HPRT1 and GUSB by GeNorm and NormFinder and HPRT1 and PPIA by BestKeeper. In retinal endothelial cells the most stable genes ranked were TBP and PGK1 by GeNorm and NormFinder and HPRT1 by BestKeeper. The least stable gene for both cell types was 18S with all 3 algorithms. Conclusions We have identified the most stable endogenous control genes in intraocular endothelial cells. It is suggested future qRT-PCR studies using these cells would benefit from adopting the genes identified in this study as the most appropriate endogenous control genes.

  6. Safeguarding nonhuman primate iPS cells with suicide genes.

    Science.gov (United States)

    Zhong, Bonan; Watts, Korashon L; Gori, Jennifer L; Wohlfahrt, Martin E; Enssle, Joerg; Adair, Jennifer E; Kiem, Hans-Peter

    2011-09-01

    The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, resulting in the formation of teratomas, have remained considerable obstacles. Here, we demonstrate the utility of suicide genes to safeguard iPS cells and their derivatives. We found suicide genes can control the cell fate of iPS cells in vitro and in vivo without interfering with their pluripotency and self-renewal capacity. This study will be useful to evaluate the safety of iPS cell technology in a clinically highly relevant, large animal model and further benefit the clinical use of human iPS cells.

  7. Stem Leydig cell differentiation: gene expression during development of the adult rat population of Leydig cells.

    Science.gov (United States)

    Stanley, Erin L; Johnston, Daniel S; Fan, Jinjiang; Papadopoulos, Vassilios; Chen, Haolin; Ge, Ren-Shan; Zirkin, Barry R; Jelinsky, Scott A

    2011-12-01

    Leydig cells are the testosterone-producing cells in the adult male. Adult Leydig cells (ALCs) develop from stem Leydig cells (SLCs) through at least two intermediate cells, progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). Microarray gene expression was used to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to that in a well-established stem cell, bone marrow stem cells (BSCs). We show that the pattern of gene expression by SLCs was more similar to the expression by BSCs, an established stem cell outside the male reproductive tract, than to any of the cells in the Leydig cell developmental lineage. These results indicated that the SLCs have many of the molecular characteristics of other stem cells. Pathway analysis indicated that development of Leydig cells from SLCs to PLCs was associated with decreased expression of genes related to adhesion and increased expression of genes related to steroidogenesis. Gene expression changes between PLCs and ILCs and between ILCs and ALCs were relatively minimal, suggesting that these cells are highly similar. In contrast, gene expression changes between SLCs and ALCs were quite distinct.

  8. Patterns of expression of cell wall related genes in sugarcane

    Directory of Open Access Journals (Sweden)

    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  9. Experimental gene therapy using p21Waf1 gene for esophageal squamous cell carcinoma by gene gun technology.

    Science.gov (United States)

    Tanaka, Yuichi; Fujii, Teruhiko; Yamana, Hideaki; Kato, Seiya; Morimatsu, Minoru; Shirouzu, Kazuo

    2004-10-01

    In our previous study, the proliferation rate of esophageal squamous cell carcinoma cell lines, which poorly expressed p21Waf1, was found to be regulated by p21Waf1 gene transfection using adenovirus vector. In the present study, in order to examine the effect of p21Waf1 gene therapy in esophageal cancer, we used gene gun technology, which proved to be a powerful method to introduce the p21Waf1 gene into esophageal cancer cells. p21Waf1 transfection to KE3 and YES2 cells (weakly expressed p21Waf1 protein cells) showed a high expression of p21Waf1 protein after applying this gene gun technique. In KE3 and YES2 cells, statistical significant growth inhibition was observed after p21Waf1 transfection compared with LacZ transfection (KE3, p=0.0009; YES2, pgun technique significantly inhibited the low basal p21Waf1 expressed esophageal cancer cell growth in vitro and in vivo. Furthermore, p21Waf1 transfection strongly enhanced the effect of 5Fu suggesting that p21Waf1 may prove beneficial in chemotherapy combined with gene therapy using gene gun technology in patients with esophageal cancer who have a low level of p21Waf1 expressed tumor.

  10. The ancestral gene repertoire of animal stem cells.

    Science.gov (United States)

    Alié, Alexandre; Hayashi, Tetsutaro; Sugimura, Itsuro; Manuel, Michaël; Sugano, Wakana; Mano, Akira; Satoh, Nori; Agata, Kiyokazu; Funayama, Noriko

    2015-12-22

    Stem cells are pivotal for development and tissue homeostasis of multicellular animals, and the quest for a gene toolkit associated with the emergence of stem cells in a common ancestor of all metazoans remains a major challenge for evolutionary biology. We reconstructed the conserved gene repertoire of animal stem cells by transcriptomic profiling of totipotent archeocytes in the demosponge Ephydatia fluviatilis and by tracing shared molecular signatures with flatworm and Hydra stem cells. Phylostratigraphy analyses indicated that most of these stem-cell genes predate animal origin, with only few metazoan innovations, notably including several partners of the Piwi machinery known to promote genome stability. The ancestral stem-cell transcriptome is strikingly poor in transcription factors. Instead, it is rich in RNA regulatory actors, including components of the "germ-line multipotency program" and many RNA-binding proteins known as critical regulators of mammalian embryonic stem cells.

  11. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Anna Oliva

    2005-07-01

    Full Text Available Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast. The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  12. Expression of HOX C homeobox genes in lymphoid cells.

    Science.gov (United States)

    Lawrence, H J; Stage, K M; Mathews, C H; Detmer, K; Scibienski, R; MacKenzie, M; Migliaccio, E; Boncinelli, E; Largman, C

    1993-08-01

    The class I homeobox genes located in four clusters in mammalian genomes (HOX A, HOX B, HOX C, and HOX D) appear to play a major role in fetal development. Previous surveys of homeobox gene expression in human leukemic cell lines have shown that certain HOX A genes are expressed only in myeloid cell lines, whereas HOX B gene expression is largely restricted to cells with erythroid potential. We now report a survey of the expression patterns of 9 homeobox genes from the HOX C locus in a panel of 24 human and 7 murine leukemic cell lines. The most striking observation is the lymphoid-specific pattern of expression of HOX C4, located at the 3' end of the locus. A major transcript of 1.9 kilobases is observed in both T-cell and B-cell lines. HOX C4 expression is also detected in normal human marrow and peripheral blood lymphocytes, but not in mature granulocytes or monocytes. HOX C8 is also expressed in human lymphoid cells but is expressed in other blood cell types as well. However, the HOX C8 transcript pattern is lineage specific. These data, in conjunction with earlier findings, suggest that homeobox gene expression influences lineage determination during hematopoiesis.

  13. Genome Binding and Gene Regulation by Stem Cell Transcription Factors

    NARCIS (Netherlands)

    J.H. Brandsma (Johan)

    2016-01-01

    markdownabstractNearly all cells of an individual organism contain the same genome. However, each cell type transcribes a different set of genes due to the presence of different sets of cell type-specific transcription factors. Such transcription factors bind to regulatory regions such as promoters

  14. Engineering T cell immunity by TCR gene transfer

    NARCIS (Netherlands)

    Linnemann, Carsten

    2013-01-01

    T cell responses against tumor-antigens are frequently observed for some human malignancies, in particular melanoma. However, the spontaneous development of T cell responses of a sufficient strength to eradicate human malignancies is rare. The transfer of T cell receptor (TCR) αβ genes into autologo

  15. Gastrin gene expression and regulation in rat islet cell lines.

    Science.gov (United States)

    Brand, S J; Wang, T C

    1988-11-15

    Gastrin gene expression was observed in two permanent rat insulinoma (RIN) cell lines derived from a rat insulinoma. Gastrin expression was selective; highest expression was seen in a cell line which did not express other islet cell hormones. Gastrin mRNA transcription initiated from the same promoter as antral gastrin mRNA. DNA transfection studies with a gastrin chloramphenicol acetyltransferase chimeric gene showed higher expression in gastrin-expressing RIN cells than non-gastrin-expressing islet cells. This implies that gastrin-expressing RIN cells selectively express a trans-acting transcriptional activator which binds to cis-acting regulatory sequences within the 5'-flanking DNA sequence and first exon of the gastrin gene. The gastrin peptide precursor synthesized in these RIN cell lines is subject to the same repertoire of posttranslational modifications within the cell's secretory apparatus (endoproteolytic cleavage, tyrosine sulfation, and C-terminal amidation) as seen in antral G cells. Gastrin mRNA levels in these RIN cells were selectively increased by increasing the extracellular calcium concentration. Membrane depolarization also stimulated gastrin mRNA levels, probably through activation of voltage-sensitive calcium channels. Thus, these gastrin-expressing RIN cell lines provide permanent cell lines useful in analyzing the cellular regulation of gastrin gene expression.

  16. Gene-modified hematopoietic stem cells for cancer immunotherapy.

    Science.gov (United States)

    Larson, Sarah; De Oliveira, Satiro N

    2014-01-01

    The rapid expansion of available cancer immunotherapies has resulted in favorable early outcomes. Specifically the use of gene therapy to introduce chimeric antigen receptors (CARs) and T cell receptors (TCRs) in T cells creates new immunotherapy options for patients. While showing early success with these approaches, limitations remain that can be overcome by the use of modification of hematopoietic stem cells (HSCs) to express CARs and TCRs. With modern gene therapy technologies, increased safety and control of the modification of the HSCs can be achieved through the use of a suicide gene.

  17. The human T cell receptor alpha variable (TRAV) genes.

    Science.gov (United States)

    Scaviner, D; Lefranc, M P

    2000-01-01

    'Human T Cell Receptor Alpha Variable (TRAV) Genes', the eighth report of the 'IMGT Locus in Focus' section, comprises four tables: (1) 'Number of human germline TRAV genes at 14q11 and potential repertoire'; (2) 'Human germline TRAV genes at 14q11'; (3) 'Human TRAV allele table', and (4) 'Correspondence between the different human TRAV gene nomenclatures'. These tables are available at the IMGT Marie-Paule page of IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr:8104) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France. Copyright 2000 S. Karger AG, Basel

  18. Reference gene for primary culture of prostate cancer cells.

    Science.gov (United States)

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

    2013-04-01

    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  19. Identification of Hematopoietic Stem Cell Engraftment Genes in Gene Therapy Studies.

    Science.gov (United States)

    Powers, John M; Trobridge, Grant D

    2013-09-01

    Hematopoietic stem cell (HSC) therapy using replication-incompetent retroviral vectors is a promising approach to provide life-long correction for genetic defects. HSC gene therapy clinical studies have resulted in functional cures for several diseases, but in some studies clonal expansion or leukemia has occurred. This is due to the dyregulation of endogenous host gene expression from vector provirus insertional mutagenesis. Insertional mutagenesis screens using replicating retroviruses have been used extensively to identify genes that influence oncogenesis. However, retroviral mutagenesis screens can also be used to determine the role of genes in biological processes such as stem cell engraftment. The aim of this review is to describe the potential for vector insertion site data from gene therapy studies to provide novel insights into mechanisms of HSC engraftment. In HSC gene therapy studies dysregulation of host genes by replication-incompetent vector proviruses may lead to enrichment of repopulating clones with vector integrants near genes that influence engraftment. Thus, data from HSC gene therapy studies can be used to identify novel candidate engraftment genes. As HSC gene therapy use continues to expand, the vector insertion site data collected will be of great interest to help identify novel engraftment genes and may ultimately lead to new therapies to improve engraftment.

  20. Regulation of cell-to-cell variability in divergent gene expression

    Science.gov (United States)

    Yan, Chao; Wu, Shuyang; Pocetti, Christopher; Bai, Lu

    2016-03-01

    Cell-to-cell variability (noise) is an important feature of gene expression that impacts cell fitness and development. The regulatory mechanism of this variability is not fully understood. Here we investigate the effect on gene expression noise in divergent gene pairs (DGPs). We generated reporters driven by divergent promoters, rearranged their gene order, and probed their expressions using time-lapse fluorescence microscopy and single-molecule fluorescence in situ hybridization (smFISH). We show that two genes in a co-regulated DGP have higher expression covariance compared with the separate, tandem and convergent configurations, and this higher covariance is caused by more synchronized firing of the divergent transcriptions. For differentially regulated DGPs, the regulatory signal of one gene can stochastically `leak' to the other, causing increased gene expression noise. We propose that the DGPs' function in limiting or promoting gene expression noise may enhance or compromise cell fitness, providing an explanation for the conservation pattern of DGPs.

  1. Undifferentiated pulp cells and odontoblast-like cells share genes involved in the process of odontogenesis.

    Science.gov (United States)

    Ferreira, Maidy Rehder Wimmers; Dernowsek, Janaína; Passos, Geraldo A; Bombonato-Prado, Karina Fittipaldi

    2015-04-01

    Expression of a large number of genes during differentiation of undifferentiated pulp cells into odontoblastic cells is still unknown, hence the aim of this investigation was to compare undifferentiated pulp cells (OD-21) and odontoblast-like cells (MDPC-23) through the assessment of cell stimulation and gene expression profiling. The cells were cultured and after the experimental periods, there were evaluated cell proliferation and viability as well as alkaline phosphatase activity (ALP) and mineralization nodules. To evaluate gene expression it was used fluorescence cDNA microarray technology in addition to bioinformatics programmes such as SAM (significance analysis of microarrays). Gene expression was validated by Real Time PCR (qPCR). The results showed that viability was above 80% in both cells, cell proliferation and ALP activity was higher in MDPC-23 cells and mineralization nodules were present only in the cultures of odontoblast-like cells. There were observed genes associated to odontogenesis with similar behaviour in both cell types, such as Il10, Traf6, Lef1 and Hspa8. Regions of the heatmap showed differences in induction and repression of genes such as Jak2 and Fas. OD-21 cells share many genes with similar behaviour to MDPC-23 cells, suggesting their potential to differentiate into odontoblasts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Evolution vs the number of gene copies per primitive cell.

    Science.gov (United States)

    Koch, A L

    1984-01-01

    Computer simulations are presented of the rate at which an advantageous mutant would displace the prototype in a replicating system without an accurate segregation mechanism. If the number of gene copies in the system is indefinitely large, Darwinian evolution is essentially stopped because there is no coupling of phenotype with genotype, i.e., there is no growth advantage to the advantageous gene relative to the prototype and therefore no "survival of the fittest." The inhibition of evolution due to a number of gene copies less than 100 would have been not insurmountable. Although the presence of multiple copies would have allowed replacement by an advantageous mutant, it provided a way for the primitive cell to conserve less immediately useful genes that could evolve into different or more effective genes. This possibility was lost as accurate segregation mechanisms evolved and cells with few copies of each gene, such as modern procaryotes, arose.

  3. Gene Therapy In Squamous Cell Carcinoma – A Short Review

    Directory of Open Access Journals (Sweden)

    Soma Susan Varghese

    2011-07-01

    Full Text Available Oral cancer remains one of the leading causes of death world wide. Various means to destroy tumor cells preferentially have been developed; gene therapy is one among them with less treatment morbidity. Gene therapy involves the transfer of therapeutic or working copy of genes into a specific cell of an individual in order to repair a faulty copy of gene. The alteration can be accomplished by repairing or replacing the damaged DNA by various strategies and vectors. To date genetically altered viruses are commonly used as gene delivery vehicle (vector which has an advantage of evolutionary selection of host-virus relation. Non viral vectors which include the physical transfection of genes can be accomplished by electrophoration, microinjection, or use of ballistic particles and chemical transfection by forming liposomes.

  4. A functional profile of gene expression in ARPE-19 cells

    Directory of Open Access Journals (Sweden)

    Johnson Dianna A

    2005-11-01

    Full Text Available Abstract Background Retinal pigment epithelium cells play an important role in the pathogenesis of age related macular degeneration. Their morphological, molecular and functional phenotype changes in response to various stresses. Functional profiling of genes can provide useful information about the physiological state of cells and how this state changes in response to disease or treatment. In this study, we have constructed a functional profile of the genes expressed by the ARPE-19 cell line of retinal pigment epithelium. Methods Using Affymetrix MAS 5.0 microarray analysis, genes expressed by ARPE-19 cells were identified. Using GeneChip® annotations, these genes were classified according to their known functions to generate a functional gene expression profile. Results We have determined that of approximately 19,044 unique gene sequences represented on the HG-U133A GeneChip® , 6,438 were expressed in ARPE-19 cells irrespective of the substrate on which they were grown (plastic, fibronectin, collagen, or Matrigel. Rather than focus our subsequent analysis on the identity or level of expression of each individual gene in this large data set, we examined the number of genes expressed within 130 functional categories. These categories were selected from a library of HG-U133A GeneChip® annotations linked to the Affymetrix MAS 5.0 data sets. Using this functional classification scheme, we were able to categorize about 70% of the expressed genes and condense the original data set of over 6,000 data points into a format with 130 data points. The resulting ARPE-19 Functional Gene Expression Profile is displayed as a percentage of ARPE-19-expressed genes. Conclusion The Profile can readily be compared with equivalent microarray data from other appropriate samples in order to highlight cell-specific attributes or treatment-induced changes in gene expression. The usefulness of these analyses is based on the assumption that the numbers of genes

  5. Cancer genes hypermethylated in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Vincenzo Calvanese

    Full Text Available Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

  6. Lab-specific gene expression signatures in pluripotent stem cells.

    Science.gov (United States)

    Newman, Aaron M; Cooper, James B

    2010-08-06

    Pluripotent stem cells derived from both embryonic and reprogrammed somatic cells have significant potential for human regenerative medicine. Despite similarities in developmental potential, however, several groups have found fundamental differences between embryonic stem cell (ESC) and induced-pluripotent stem cell (iPSC) lines that may have important implications for iPSC-based medical therapies. Using an unsupervised clustering algorithm, we further studied the genetic homogeneity of iPSC and ESC lines by reanalyzing microarray gene expression data from seven different laboratories. Unexpectedly, this analysis revealed a strong correlation between gene expression signatures and specific laboratories in both ESC and iPSC lines. Nearly one-third of the genes with lab-specific expression signatures are also differentially expressed between ESCs and iPSCs. These data are consistent with the hypothesis that in vitro microenvironmental context differentially impacts the gene expression signatures of both iPSCs and ESCs.

  7. Effects of cell cycle noise on excitable gene circuits

    CERN Document Server

    Veliz-Cuba, Alan; Bennett, Matthew R; Josić, Krešimir; Ott, William

    2016-01-01

    We assess the impact of cell cycle noise on gene circuit dynamics. For bistable genetic switches and excitable circuits, we find that transitions between metastable states most likely occur just after cell division and that this concentration effect intensifies in the presence of transcriptional delay. We explain this concentration effect with a 3-states stochastic model. For genetic oscillators, we quantify the temporal correlations between daughter cells induced by cell division. Temporal correlations must be captured properly in order to accurately quantify noise sources within gene networks.

  8. Gene and stem cell therapy of the hair follicle.

    Science.gov (United States)

    Hoffman, Robert M

    2005-01-01

    The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is discussed. Hair follicle delivery systems are described such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

  9. The mechanism of gene targeting in human somatic cells.

    Directory of Open Access Journals (Sweden)

    Yinan Kan

    2014-04-01

    Full Text Available Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB repair known as homologous recombination (HR. The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

  10. The mechanism of gene targeting in human somatic cells.

    Science.gov (United States)

    Kan, Yinan; Ruis, Brian; Lin, Sherry; Hendrickson, Eric A

    2014-04-01

    Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.

  11. Development of gene and stem cell therapy for ocular neurodegeneration

    Institute of Scientific and Technical Information of China (English)

    Jing-Xue; Zhang; Ning-Li; Wang; Qing-Jun; Lu

    2015-01-01

    Retinal degenerative diseases pose a serious threat to eye health, but there is currently no effective treatment available. Recent years have witnessed rapid development of several cutting-edge technologies, such as gene therapy, stem cell therapy, and tissue engineering. Due to the special features of ocular structure, some of these technologies have been translated into ophthalmological clinic practice with fruitful achievements, setting a good example for other fields. This paper reviews the development of the gene and stem cell therapies in ophthalmology.

  12. Stromal mesenchyme cell genes of the human prostate and bladder

    Directory of Open Access Journals (Sweden)

    Pascal Laura E

    2005-12-01

    Full Text Available Abstract Background Stromal mesenchyme cells play an important role in epithelial differentiation and likely in cancer as well. Induction of epithelial differentiation is organ-specific, and the genes responsible could be identified through a comparative genomic analysis of the stromal cells from two different organs. These genes might be aberrantly expressed in cancer since cancer could be viewed as due to a defect in stromal signaling. We propose to identify the prostate stromal genes by analysis of differentially expressed genes between prostate and bladder stromal cells, and to examine their expression in prostate cancer. Methods Immunohistochemistry using antibodies to cluster designation (CD cell surface antigens was first used to characterize the stromas of the prostate and bladder. Stromal cells were prepared from either prostate or bladder tissue for cell culture. RNA was isolated from the cultured cells and analyzed by DNA microarrays. Expression of candidate genes in normal prostate and prostate cancer was examined by RT-PCR. Results The bladder stroma was phenotypically different from that of the prostate. Most notable was the presence of a layer of CD13+ cells adjacent to the urothelium. This structural feature was also seen in the mouse bladder. The prostate stroma was uniformly CD13-. A number of differentially expressed genes between prostate and bladder stromal cells were identified. One prostate gene, proenkephalin (PENK, was of interest because it encodes a hormone. Secreted proteins such as hormones and bioactive peptides are known to mediate cell-cell signaling. Prostate stromal expression of PENK was verified by an antibody raised against a PENK peptide, by RT-PCR analysis of laser-capture microdissected stromal cells, and by database analysis. Gene expression analysis showed that PENK expression was down-regulated in prostate cancer. Conclusion Our findings show that the histologically similar stromas of the prostate and

  13. Generation of antigen-specific T cell immunity through T cell receptor gene transfer

    NARCIS (Netherlands)

    Coccoris, Miriam

    2009-01-01

    Cancer cells often escape the attack of immune cells because they originate from self-tissue. Through T cell receptor gene transfer it is possible to equip peripheral T cells with a desired specificity, and this strategy may be useful to generate tumor-specific T cells for the treatment of cancer in

  14. Generation of antigen-specific T cell immunity through T cell receptor gene transfer

    NARCIS (Netherlands)

    Coccoris, Miriam

    2009-01-01

    Cancer cells often escape the attack of immune cells because they originate from self-tissue. Through T cell receptor gene transfer it is possible to equip peripheral T cells with a desired specificity, and this strategy may be useful to generate tumor-specific T cells for the treatment of cancer in

  15. Cell- and gene-based approaches to tendon regeneration.

    Science.gov (United States)

    Nixon, Alan J; Watts, Ashlee E; Schnabel, Lauren V

    2012-02-01

    Repair of rotator cuff tears in experimental models has been significantly improved by the use of enhanced biologic approaches, including platelet-rich plasma, bone marrow aspirate, growth factor supplements, and cell- and gene-modified cell therapy. Despite added complexity, cell-based therapies form an important part of enhanced repair, and combinations of carrier vehicles, growth factors, and implanted cells provide the best opportunity for robust repair. Bone marrow-derived mesenchymal stem cells provide a stimulus for repair in flexor tendons, but application in rotator cuff repair has not shown universally positive results. The use of scaffolds such as platelet-rich plasma, fibrin, and synthetic vehicles and the use of gene priming for stem cell differentiation and local anabolic and anti-inflammatory impact have both provided essential components for enhanced tendon and tendon-to-bone repair in rotator cuff disruption. Application of these research techniques in human rotator cuff injury has generally been limited to autologous platelet-rich plasma, bone marrow concentrate, or bone marrow aspirates combined with scaffold materials. Cultured mesenchymal progenitor therapy and gene-enhanced function have not yet reached clinical trials in humans. Research in several animal species indicates that the concept of gene-primed stem cells, particularly embryonic stem cells, combined with effective culture conditions, transduction with long-term integrating vectors carrying anabolic growth factors, and development of cells conditioned by use of RNA interference gene therapy to resist matrix metalloproteinase degradation, may constitute potential advances in rotator cuff repair. This review summarizes cell- and gene-enhanced cell research for tendon repair and provides future directions for rotator cuff repair using biologic composites.

  16. Stem Cell Based Gene Therapy in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Jae Heon Kim

    2014-01-01

    Full Text Available Current prostate cancer treatment, especially hormone refractory cancer, may create profound iatrogenic outcomes because of the adverse effects of cytotoxic agents. Suicide gene therapy has been investigated for the substitute modality for current chemotherapy because it enables the treatment targeting the cancer cells. However the classic suicide gene therapy has several profound side effects, including immune-compromised due to viral vector. Recently, stem cells have been regarded as a new upgraded cellular vehicle or vector because of its homing effects. Suicide gene therapy using genetically engineered mesenchymal stem cells or neural stem cells has the advantage of being safe, because prodrug administration not only eliminates tumor cells but consequently kills the more resistant therapeutic stem cells as well. The attractiveness of prodrug cancer gene therapy by stem cells targeted to tumors lies in activating the prodrug directly within the tumor mass, thus avoiding systemic toxicity. Therapeutic achievements using stem cells in prostate cancer include the cytosine deaminase/5-fluorocytosine prodrug system, herpes simplex virus thymidine kinase/ganciclovir, carboxyl esterase/CPT11, and interferon-beta. The aim of this study is to review the stem cell therapy in prostate cancer including its proven mechanisms and also limitations.

  17. Gene expression of manganese superoxide dismutase in human glioma cells

    Directory of Open Access Journals (Sweden)

    Novi S. Hardiany

    2010-02-01

    Full Text Available Aim This study analyze the MnSOD gene expression as endogenous antioxidant in human glioma cells compared with leucocyte cells as control.Methods MnSOD gene expression of 20 glioma patients was analyzed by measuring the relative expression of mRNA and enzyme activity of MnSOD in brain and leucocyte cells. The relative expression of mRNA MnSOD was determined by using quantitative Real Time RT-PCR and the enzyme activity of MnSOD using biochemical kit assay (xantine oxidase inhibition. Statistic analysis for mRNA and enzyme activity of MnSOD was performed using Kruskal Wallis test.Results mRNA of MnSOD in glioma cells of 70% sample was 0.015–0.627 lower, 10% was 1.002-1.059 and 20% was 1.409-6.915 higher than in leucocyte cells. Also the specific activity of MnSOD enzyme in glioma cells of 80% sample showed 0,064-0,506 lower and 20% sample was 1.249-2.718 higher than in leucocyte cells.Conclusion MnSOD gene expression in human glioma cells are significantly lower than its expression in leucocytes cells. (Med J Indones 2010; 19:21-5Keywords : MnSOD, glioma, gene expression

  18. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  19. Impact of methoxyacetic acid on mouse Leydig cell gene expression

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    Waxman David J

    2010-06-01

    Full Text Available Abstract Background Methoxyacetic acid (MAA is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Methods Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling. Results A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling. Conclusions These findings

  20. Engineering Synthetic Gene Circuits in Living Cells with CRISPR Technology.

    Science.gov (United States)

    Jusiak, Barbara; Cleto, Sara; Perez-Piñera, Pablo; Lu, Timothy K

    2016-07-01

    One of the goals of synthetic biology is to build regulatory circuits that control cell behavior, for both basic research purposes and biomedical applications. The ability to build transcriptional regulatory devices depends on the availability of programmable, sequence-specific, and effective synthetic transcription factors (TFs). The prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR) system, recently harnessed for transcriptional regulation in various heterologous host cells, offers unprecedented ease in designing synthetic TFs. We review how CRISPR can be used to build synthetic gene circuits and discuss recent advances in CRISPR-mediated gene regulation that offer the potential to build increasingly complex, programmable, and efficient gene circuits in the future.

  1. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  2. Screening for the Most Suitable Reference Genes for Gene Expression Studies in Equine Milk Somatic Cells.

    Directory of Open Access Journals (Sweden)

    Jakub Cieslak

    Full Text Available Apart from the well-known role of somatic cell count as a parameter reflecting the inflammatory status of the mammary gland, the composition of cells isolated from milk is considered as a valuable material for gene expression studies in mammals. Due to its unique composition, in recent years an increasing interest in mare's milk consumption has been observed. Thus, investigating the genetic background of horse's milk variability presents and interesting study model. Relying on 39 milk samples collected from mares representing three breeds (Polish Primitive Horse, Polish Cold-blooded Horse, Polish Warmblood Horse we aimed to investigate the utility of equine milk somatic cells as a source of mRNA and to screen the best reference genes for RT-qPCR using geNorm and NormFinder algorithms. The results showed that despite relatively low somatic cell counts in mare's milk, the amount and the quality of the extracted RNA are sufficient for gene expression studies. The analysis of the utility of 7 potential reference genes for RT-qPCR experiments for the normalization of equine milk somatic cells revealed some differences between the outcomes of the applied algorithms, although in both cases the KRT8 and TOP2B genes were pointed as the most stable. Analysis by geNorm showed that the combination of 4 reference genes (ACTB, GAPDH, TOP2B and KRT8 is required for apropriate RT-qPCR experiments normalization, whereas NormFinder algorithm pointed the combination of KRT8 and RPS9 genes as the most suitable. The trial study of the relative transcript abundance of the beta-casein gene with the use of various types and numbers of internal control genes confirmed once again that the selection of proper reference gene combinations is crucial for the final results of each real-time PCR experiment.

  3. [Immunoglobulin genes in lymphoid cells and regulation of their transcription].

    Science.gov (United States)

    Stepchenko, A G; Urakov, D N; Luchina, N N; Deev, S M; Polianovskiĭ, O L

    1990-01-01

    The hybridoma genomes contain polyploid sets of immunoglobulin genes. We have shown, that the hybridoma PTF-02 genome contains three genes of heavy chains and two genes of light chains. The genes responsible for antibody synthesis were cloned and their structure were determined. Investigation of the kappa gene transcription and its fragments which contain regulatory sequences revealed a nuclear factor. The latter interacts with the octanucleotide localized at the promoter region of the kappa gene. The purified factor activates the transcription of the kappa gene in a heterologous cell-free system. Together with the tissue-specific factor there is also an universal factor interacting with the octanucleotide sequence. We have shown an additional factor in lymphoid cells interact with the protein which binds to the octanucleotide sequence. We have shown an additional factor in lymphoid cells interacting with the protein which binds to the octanucleotide sequence. As a result, there is a family of factors which interact with ATTTGCAT sequence. One major factor (m.w. 60 +/- 2 kDa) is an obligatory component for the initiation of immunoglobulin genes transcription.

  4. Identification of genes responsive to apoptosis in HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    Wei JIN; Le-feng QU; Ping MIN; Shan CHEN; Hong LI; He LU; Yong-tai HOU

    2004-01-01

    AIM: To identify genes responsive to apoptosis in HL-60 cells treated by homoharringtonine. METHODS: cDNA microarray technology was used to detect gene expression and the result of microarrays for genes (TIEG and VDUP1) was confirmed by Northern analysis. RESULTS: Seventy-five individual mRNAs whose mass changed significantly were identified. Among these genes (25 were up-regulated and 50 were down-regulated), most are known related to oncogenes and tumor suppressor. Some genes were involved in apoptosis signaling pathways.CONCLUSION: TGFβ and TNF apoptosis signaling pathways were initiated during apoptosis in HL-60 cells.TIEG and VDUP1 play important roles in mediating apoptosis.

  5. Binary gene induction and protein expression in individual cells

    Directory of Open Access Journals (Sweden)

    Conolly Rory B

    2006-04-01

    Full Text Available Abstract Background Eukaryotic gene transcription is believed to occur in either a binary or a graded fashion. With binary induction, a transcription activator (TA regulates the probability with which a gene template is switched from the inactive to the active state without affecting the rate at which RNA molecules are produced from the template. With graded, also called rheostat-like, induction the gene template has continuously varying levels of transcriptional activity, and the TA regulates the rate of RNA production. Support for each of these two mechanisms arises primarily from experimental studies measuring reporter proteins in individual cells, rather than from direct measurement of induction events at the gene template. Methods and results In this paper, using a computational model of stochastic gene expression, we have studied the biological and experimental conditions under which a binary induction mode operating at the gene template can give rise to differentially expressed "phenotypes" (i.e., binary, hybrid or graded at the protein level. We have also investigated whether the choice of reporter genes plays a significant role in determining the observed protein expression patterns in individual cells, given the diverse properties of commonly-used reporter genes. Our simulation confirmed early findings that the lifetimes of active/inactive promoters and half-lives of downstream mRNA/protein products are important determinants of various protein expression patterns, but showed that the induction time and the sensitivity with which the expressed genes are detected are also important experimental variables. Using parameter conditions representative of reporter genes including green fluorescence protein (GFP and β-galactosidase, we also demonstrated that graded gene expression is more likely to be observed with GFP, a longer-lived protein with low detection sensitivity. Conclusion The choice of reporter genes may determine whether protein

  6. Gene therapy for oral squamous cell carcinoma: an overview.

    Science.gov (United States)

    Saraswathi, T R; Kavitha, B; Vijayashree Priyadharsini, J

    2007-01-01

    A potential approach to the treatment of genetic disorders is gene therapy. The goal of gene therapy is to introduce therapeutic genetic material into the target cell to exert the intended therapeutic effect. Gene therapy has already shown promising results for the treatment of monogenic disorders such as severe combined immunodeficiency and haemophilia. Now the procedure has been extended to the level of treating malignant conditions such as cancer of the lungs, breast, colon etc. The prevalence of tumours of the larynx and oral cavity has increased in both developed and developing countries. This increase underscores the need for a novel therapeutic modality that would decrease or completely terminate the proliferation of malignant cells. This review highlights various types of gene therapy procedures with respect to oral squamous cell carcinoma.

  7. Gene therapy for oral squamous cell carcinoma: An overview

    Directory of Open Access Journals (Sweden)

    Saraswathi T

    2007-01-01

    Full Text Available A potential approach to the treatment of genetic disorders is gene therapy. The goal of gene therapy is to introduce therapeutic genetic material into the target cell to exert the intended therapeutic effect. Gene therapy has already shown promising results for the treatment of monogenic disorders such as severe combined immunodeficiency and haemophilia. Now the procedure has been extended to the level of treating malignant conditions such as cancer of the lungs, breast, colon etc. The prevalence of tumours of the larynx and oral cavity has increased in both developed and developing countries. This increase underscores the need for a novel therapeutic modality that would decrease or completely terminate the proliferation of malignant cells. This review highlights various types of gene therapy procedures with respect to oral squamous cell carcinoma.

  8. Transcriptional Wiring of Cell Wall-Related Genes in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Marek Mutwil; Colin Ruprecht; Federico M. Giorgi; Martin Bringmann; Bj(o)rn Usadel; Staffan Persson

    2009-01-01

    Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the correspond-ing proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of anal-yses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes (CAZy)-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface (http://aranet.mpimp-golm.mpg.de/corecarb) containing downloadable files for all the transcriptional associations.

  9. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer.

    Science.gov (United States)

    Zhang, Dingxiao; Park, Daechan; Zhong, Yi; Lu, Yue; Rycaj, Kiera; Gong, Shuai; Chen, Xin; Liu, Xin; Chao, Hsueh-Ping; Whitney, Pamela; Calhoun-Davis, Tammy; Takata, Yoko; Shen, Jianjun; Iyer, Vishwanath R; Tang, Dean G

    2016-02-29

    The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

  10. Pluripotent Stem Cells for Gene Therapy of Degenerative Muscle Diseases.

    Science.gov (United States)

    Loperfido, Mariana; Steele-Stallard, Heather B; Tedesco, Francesco Saverio; VandenDriessche, Thierry

    2015-01-01

    Human pluripotent stem cells represent a unique source for cell-based therapies and regenerative medicine. The intrinsic features of these cells such as their easy accessibility and their capacity to be expanded indefinitely overcome some limitations of conventional adult stem cells. Furthermore, the possibility to derive patient-specific induced pluripotent stem (iPS) cells in combination with the current development of gene modification methods could be used for autologous cell therapies of some genetic diseases. In particular, muscular dystrophies are considered to be a good candidate due to the lack of efficacious therapeutic treatments for patients to date, and in view of the encouraging results arising from recent preclinical studies. Some hurdles, including possible genetic instability and their efficient differentiation into muscle progenitors through vector/transgene-free methods have still to be overcome or need further optimization. Additionally, engraftment and functional contribution to muscle regeneration in pre-clinical models need to be carefully assessed before clinical translation. This review offers a summary of the advanced methods recently developed to derive muscle progenitors from pluripotent stem cells, as well as gene therapy by gene addition and gene editing methods using ZFNs, TALENs or CRISPR/Cas9. We have also discussed the main issues that need to be addressed for successful clinical translation of genetically corrected patient-specific pluripotent stem cells in autologous transplantation trials for skeletal muscle disorders.

  11. Experiments on Gene Transferring to Primary Hematopoietic Cells by Liposome

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Liposomes have showed many advantages in mediating exogenous gene into many cell types in vitro and in vivo. But few data are available concerning gene transfer into hematopoietic cells. In this report, we described two-marker genes (Neo R and Lac Z) co-transferred into hematopoietic cells of human and mouse by using liposome in vitro. The efficiency of gene transfer was tested by Xgal staining and observation of colony formation. The X-gal blue staining rate of transduced cells was about (13.33±2. 68) % in human and about (16. 28±2.95) % in mouse without G418 selection. After G418 selection, the blue cell rate was (46. 06±3.47)%in human and (43. 45±4. 1) % in mouse, which were markedly higher than those before selection, suggesting that high-efficiency gene transfer and expression could be attained in primary hematopoietic cells using this easy and harmless transduction protocol. At the same time, this protocol provided experimental data for clinicians to investigate the biology of marrow reconstitution and trace the origin of relapse after autologous bone marrow transplantation for the patients with leukemia.

  12. Interdependence of cell growth and gene expression: origins and consequences.

    Science.gov (United States)

    Scott, Matthew; Gunderson, Carl W; Mateescu, Eduard M; Zhang, Zhongge; Hwa, Terence

    2010-11-19

    In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

  13. Targeted delivery of genes to endothelial cells and cell- and gene-based therapy in pulmonary vascular diseases.

    Science.gov (United States)

    Suen, Colin M; Mei, Shirley H J; Kugathasan, Lakshmi; Stewart, Duncan J

    2013-10-01

    Pulmonary arterial hypertension (PAH) is a devastating disease that, despite significant advances in medical therapies over the last several decades, continues to have an extremely poor prognosis. Gene therapy is a method to deliver therapeutic genes to replace defective or mutant genes or supplement existing cellular processes to modify disease. Over the last few decades, several viral and nonviral methods of gene therapy have been developed for preclinical PAH studies with varying degrees of efficacy. However, these gene delivery methods face challenges of immunogenicity, low transduction rates, and nonspecific targeting which have limited their translation to clinical studies. More recently, the emergence of regenerative approaches using stem and progenitor cells such as endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) have offered a new approach to gene therapy. Cell-based gene therapy is an approach that augments the therapeutic potential of EPCs and MSCs and may deliver on the promise of reversal of established PAH. These new regenerative approaches have shown tremendous potential in preclinical studies; however, large, rigorously designed clinical studies will be necessary to evaluate clinical efficacy and safety.

  14. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  15. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies that comp......It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies...... in cell adhesion and the cytoskeleton. If the proteins involved in tethering cells to the extracellular matrix are important in conferring drug resistance, it may be possible to improve chemotherapy by designing drugs that target these proteins....

  16. Gene expression disparity in giant cell tumor of bone

    Institute of Scientific and Technical Information of China (English)

    Xiaohua PAN; Shuhua YANG; Deming XIAO; Yong DAI; Lili REN

    2009-01-01

    The aim of this paper was to study the differential gene expression of giant cell tumor of bone (GCTB) by gene chip technology. Total RNA of 8 fresh GCTB specimens (Jaffe Ⅰ:6 cases, Ⅱ: 1 case, Ⅲ: 1 case; Campanacci Ⅰ:6 cases, Ⅱ:1 case, Ⅲ:1 case; Enneking Staging G0T1-2M0, 5 cases, G1T1-2M0: 2 cases, G1T2M0: 1 case) and 4 normal bony callus specimens (the control group) were extracted and purified to get mRNA and then reverse transcribed to complementary DNA, respectively. Microarray screening with a set of 8064 human cDNA genes was conducted to analyze the difference among the samples and the control. The hybridization signals were scanned. The gene expression disparity between the GCTB samples and normal bony callus was significantly different (P<0.01), and the disparity of over 5-fold was found in 47 genes in the GCTB specimens, with 25 genes up-regulated and 22 down-regulated including the extracellular matrix and transforming-related genes, oncogene and its homolog genes, cytokine and its receptor genes. Specific gene spectrum associated with GCTB can be identified by cDNA microarray, which will be the foundation of progressive etiology elucidation, diagnosis and treatment of GCTB.

  17. Ebola virus infection induces irregular dendritic cell gene expression.

    Science.gov (United States)

    Melanson, Vanessa R; Kalina, Warren V; Williams, Priscilla

    2015-02-01

    Filoviruses subvert the human immune system in part by infecting and replicating in dendritic cells (DCs). Using gene arrays, a phenotypic profile of filovirus infection in human monocyte-derived DCs was assessed. Monocytes from human donors were cultured in GM-CSF and IL-4 and were infected with Ebola virus Kikwit variant for up to 48 h. Extracted DC RNA was analyzed on SuperArray's Dendritic and Antigen Presenting Cell Oligo GEArray and compared to uninfected controls. Infected DCs exhibited increased expression of cytokine, chemokine, antiviral, and anti-apoptotic genes not seen in uninfected controls. Significant increases of intracellular antiviral and MHC I and II genes were also noted in EBOV-infected DCs. However, infected DCs failed to show any significant difference in co-stimulatory T-cell gene expression from uninfected DCs. Moreover, several chemokine genes were activated, but there was sparse expression of chemokine receptors that enabled activated DCs to home to lymph nodes. Overall, statistically significant expression of several intracellular antiviral genes was noted, which may limit viral load but fails to stop replication. EBOV gene expression profiling is of vital importance in understanding pathogenesis and devising novel therapeutic treatments such as small-molecule inhibitors.

  18. Cancer specificity of promoters of the genes controlling cell proliferation.

    Science.gov (United States)

    Kashkin, Kirill; Chernov, Igor; Stukacheva, Elena; Monastyrskaya, Galina; Uspenskaya, Natalya; Kopantzev, Eugene; Sverdlov, Eugene

    2015-02-01

    Violation of proliferation control is a common feature of cancer cells. We put forward the hypothesis that promoters of genes involved in the control of cell proliferation should possess intrinsic cancer specific activity. We cloned promoter regions of CDC6, POLD1, CKS1B, MCM2, and PLK1 genes into pGL3 reporter vector and studied their ability to drive heterologous gene expression in transfected cancer cells of different origin and in normal human fibroblasts. Each promoter was cloned in short (335-800 bp) and long (up to 2.3 kb) variants to cover probable location of core and whole promoter regulatory elements. Cloned promoters were significantly more active in cancer cells than in normal fibroblasts that may indicate their cancer specificity. Both versions of CDC6 promoters were shown to be most active while the activities of others were close to that of BIRC5 gene (survivin) gene promoter. Long and short variants of each cloned promoter demonstrated very similar cancer specificity with the exception of PLK1-long promoter that was substantially more specific than its short variant and other promoters under study. The data indicate that most of the important cis-regulatory transcription elements responsible for intrinsic cancer specificity are located in short variants of the promoters under study. CDC6 short promoter may serve as a promising candidate for transcription targeted cancer gene therapy.

  19. Nucleus- and cell-specific gene expression in monkey thalamus.

    Science.gov (United States)

    Murray, Karl D; Choudary, Prabhakara V; Jones, Edward G

    2007-02-06

    Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input-output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys, we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative PCR and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathways Analysis revealed overrepresentation of GO categories related to development, morphogenesis, cell-cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes, many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons; a calmodulin-binding protein PCP4; the bone extracellular matrix molecules SPP1 and SPARC; and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype, and connectivity during development and their maintenance in the adult thalamus.

  20. Positron emission tomography reporter genes and reporter probes: gene and cell therapy applications.

    Science.gov (United States)

    Yaghoubi, Shahriar S; Campbell, Dean O; Radu, Caius G; Czernin, Johannes

    2012-01-01

    Positron emission tomography (PET) imaging reporter genes (IRGs) and PET reporter probes (PRPs) are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.

  1. Positron Emission Tomography Reporter Genes and Reporter Probes: Gene and Cell Therapy Applications

    Directory of Open Access Journals (Sweden)

    Shahriar S. Yaghoubi, Dean O. Campbell, Caius G. Radu, Johannes Czernin

    2012-01-01

    Full Text Available Positron emission tomography (PET imaging reporter genes (IRGs and PET reporter probes (PRPs are amongst the most valuable tools for gene and cell therapy. PET IRGs/PRPs can be used to non-invasively monitor all aspects of the kinetics of therapeutic transgenes and cells in all types of living mammals. This technology is generalizable and can allow long-term kinetics monitoring. In gene therapy, PET IRGs/PRPs can be used for whole-body imaging of therapeutic transgene expression, monitoring variations in the magnitude of transgene expression over time. In cell or cellular gene therapy, PET IRGs/PRPs can be used for whole-body monitoring of therapeutic cell locations, quantity at all locations, survival and proliferation over time and also possibly changes in characteristics or function over time. In this review, we have classified PET IRGs/PRPs into two groups based on the source from which they were derived: human or non-human. This classification addresses the important concern of potential immunogenicity in humans, which is important for expansion of PET IRG imaging in clinical trials. We have then discussed the application of this technology in gene/cell therapy and described its use in these fields, including a summary of using PET IRGs/PRPs in gene and cell therapy clinical trials. This review concludes with a discussion of the future direction of PET IRGs/PRPs and recommends cell and gene therapists collaborate with molecular imaging experts early in their investigations to choose a PET IRG/PRP system suitable for progression into clinical trials.

  2. Expression and function of FERMT genes in colon carcinoma cells.

    Science.gov (United States)

    Kiriyama, Kenji; Hirohashi, Yoshihiko; Torigoe, Toshihiko; Kubo, Terufumi; Tamura, Yasuaki; Kanaseki, Takayuki; Takahashi, Akari; Nakazawa, Emiri; Saka, Eri; Ragnarsson, Charlotte; Nakatsugawa, Munehide; Inoda, Satoko; Asanuma, Hiroko; Takasu, Hideo; Hasegawa, Tadashi; Yasoshima, Takahiro; Hirata, Koichi; Sato, Noriyuki

    2013-01-01

    Invasion into the matrix is one of hallmarks of malignant diseases and is the first step for tumor metastasis. Thus, analysis of the molecular mechanisms of invasion is essential to overcome tumor cell invasion. In the present study, we screened for colon carcinoma-specific genes using a cDNA microarray database of colon carcinoma tissues and normal colon tissues, and we found that fermitin family member-1 (FERMT1) is overexpressed in colon carcinoma cells. FRRMT1, FERMT2 and FERMT3 expression was investigated in colon carcinoma cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that only FERMT1 had cancer cell-specific expression. Protein expression of FERMT1 was confirmed by western blotting and immunohistochemical staining. To address the molecular functions of FERMT genes in colon carcinoma cells, we established FERMT1-, FERMT2- and FERMT3-overexpressing colon carcinoma cells. FERMT1-overexpressing cells exhibited greater invasive ability than did FERMT2- and FERMT3-overexpressing cells. On the other hand, FERMT1-, FERMT2- and FERMT3-overexpressing cells exhibited enhancement of cell growth. Taken together, the results of this study indicate that FERMT1 is expressed specifically in colon carcinoma cells, and has roles in matrix invasion and cell growth. These findings indicate that FERMT1 is a potential molecular target for cancer therapy.

  3. [Expression of rice dwarf virus outer coat protein gene(S8) in insect cells].

    Science.gov (United States)

    Li, S; Liu, H; Chen, Z; Li, Y

    2001-04-01

    Outer coat protein gene(S8) of RDV was cloned into the transfer vector pVL 1393 to construct a recombinant vector pVL1393-S8. The recombinant vector pVL1393-S8 and the linear baculovirus RP23. LacZ were cotransfected into sf9 cells to produce the recombinant virus RP23-S8. RP23-S8 infected sf9 cells were collected and analysed by SDS-PAGE and Western-blot. The results showed that the S8 gene of RDV was expressed in sf9 cells and the expression level of sf9 cells was higher between 72-96 h after infected.

  4. [Differentiation of functional cells from iPS cells by efficient gene transfer].

    Science.gov (United States)

    Kawabata, Kenji; Tashiro, Katsuhisa; Mizuguchi, Hiroyuki

    2010-11-01

    Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. By using an adenovirus (Ad) vector containing the cytomegalovirus enhancer/beta-actin (CA) promoters, we have developed an efficient transduction system for mouse mesenchymal stem cells and embryonic stem (ES) cells. Also, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector could efficiently transduce transgenes into mouse iPS cells. We found that the CA promoter had potent transduction ability in iPS cells. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to promote cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.

  5. Suicide genes: monitoring cells in patients with a safety switch.

    Science.gov (United States)

    Eissenberg, Linda G; Rettig, Michael; Dehdashti, Farrokh; Piwnica-Worms, David; DiPersio, John F

    2014-01-01

    Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical trial used (18)F-9-(4-fluoro-3-hydroxymethylbutyl)guanine ((18)FHBG) and positron emission tomography (PET)/CT scans to follow T cells transduced with herpes simplex virus thymidine kinase after administration to patients. Guided by preclinical data we ultimately hope to discern whether a particular pattern of transduced T cell migration within patients reflects early development of graft vs. host disease. Current difficulties in terms of choice of suicide gene, biodistribution of radiolabeled tracers in humans vs. animal models, and threshold levels of genetically modified cells needed for detection by PET/CT are discussed. As alternative suicide genes are developed, additional radiolabel probes suitable for imaging in patients should be considered.

  6. Suicide genes: monitoring cells in patients with a safety switch

    Directory of Open Access Journals (Sweden)

    Linda Groppe Eissenberg

    2014-11-01

    Full Text Available Clinical trials increasingly incorporate suicide genes either as direct lytic agents for tumors or as safety switches in therapies based on genetically modified cells. Suicide genes can also be used as non-invasive reporters to monitor the biological consequences of administering genetically modified cells to patients and gather information relevant to patient safety. These genes can monitor therapeutic outcomes addressable by early clinical intervention. As an example, our recent clinical trial used 18F-9-(4-fluoro-3-hydroxymethylbutylguanine (18FHBG and PET/CT scans to follow T cells transduced with herpes simplex virus thymidine kinase (TK after administration to patients. Guided by preclinical data we ultimately hope to discern whether a particular pattern of transduced T cell migration within patients reflects early development of Graft vs. Host Disease (GvHD. Current difficulties in terms of choice of suicide gene, biodistribution of radiolabeled tracers in humans versus animal models, and threshold levels of genetically modified cells needed for detection by PET/CT are discussed. As alternative suicide genes are developed, additional radiolabel probes suitable for imaging in patients should be considered.

  7. Gene expression profiling of chicken primordial germ cell ESTs

    Directory of Open Access Journals (Sweden)

    Lim Dajeong

    2006-08-01

    Full Text Available Abstract Background Germ cells are the only cell type that can penetrate from one generation to next generation. At the early embryonic developmental stages, germ cells originally stem from primordial germ cells, and finally differentiate into functional gametes, sperm in male or oocyte in female, after sexual maturity. This study was conducted to investigate a large-scale expressed sequence tag (EST analysis in chicken PGCs and compare the expression of the PGC ESTs with that of embryonic gonad. Results We constructed 10,851 ESTs from a chicken cDNA library of a collection of highly separated embryonic PGCs. After chimeric and problematic sequences were filtered out using the chicken genomic sequences, there were 5,093 resulting unique sequences consisting of 156 contigs and 4,937 singlets. Pearson chi-square tests of gene ontology terms in the 2nd level between PGC and embryonic gonad set showed no significance. However, digital gene expression profiling using the Audic's test showed that there were 2 genes expressed significantly with higher number of transcripts in PGCs compared with the embryonic gonads set. On the other hand, 17 genes in embryonic gonads were up-regulated higher than those in the PGC set. Conclusion Our results in this study contribute to knowledge of mining novel transcripts and genes involved in germline cell proliferation and differentiation at the early embryonic stages.

  8. Investigation of radiosensitivity gene signatures in cancer cell lines.

    Directory of Open Access Journals (Sweden)

    John S Hall

    Full Text Available Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2 by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median was investigated using Affymetrix GeneChip Exon 1.0ST (cervix or U133A Plus2 (head and neck arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4% were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI, and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

  9. In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

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    Kristiansen Glen

    2007-06-01

    Full Text Available Abstract Background Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. Results The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP and peptidylprolyl isomerase A (PPIA, all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. Conclusion Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

  10. Embryonic stem cell-derived microvesicles induce gene expression changes in Muller cells of the retina.

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    Diana Katsman

    Full Text Available Cell-derived microvesicles (MVs, recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  11. Amygdalin inhibits genes related to cell cycle in SNU-C4 human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Hae-Jeong Park; Sung-Vin Yim; Joo-Ho Chung; Seon-Pyo Hong; Seo-Hyun Yoon; Long-Shan Han; Long-Tai Zheng; Kyung-Hee Jung; Yoon-Kyung Uhm; Je-Hyun Lee; Ji-Seon Jeong; Woo-Sang Joo

    2005-01-01

    AIM: The genes were divided into seven categories according to biological function; apoptosis-reiated, immune response-related, signal transduction-related, cell cyclerelated, cell growth-related, stress response-related and transcription-related genes.METHODS: We compared the gene expression profiles of SNU-C4 cells between amygdalin-treated (5 mg/mL,24 h) and non-treated groups using cDNA microarray analysis. We selected genes downregulated in cDNA microarray and investigated mRNA levels of the genes by RT-PCR. RESULTS: Microarray showed that amygdalin downregulated especially genes belonging to cell cycle category: exonuclease 1 (EXO1), ATP-binding cassette, sub-family F, member 2 (ABCF2), MRE11 meiotic recombination 11 homolog A (MRE114), topoisomerase (DNA) I (TOP1), and FK506 binding protein 12-rapamycin-associated protein 1 (FRAP1). RT-PCR analysis revealed that mRNA levels of these genes were also decreased by amygdalin treatment in SNU-C4 human colon cancer cells.CONCLUSION: These results suggest that amygdalin have an anticancer effect via downregulation of cell cycle-related genes in SNU-C4 human colon cancer cells,and might be used for therapeutic anticancer drug.

  12. Amygdalin inhibits genes related to cell cycle in SNU-C4 human colon cancer cells.

    Science.gov (United States)

    Park, Hae-Jeong; Yoon, Seo-Hyun; Han, Long-Shan; Zheng, Long-Tai; Jung, Kyung-Hee; Uhm, Yoon-Kyung; Lee, Je-Hyun; Jeong, Ji-Seon; Joo, Woo-Sang; Yim, Sung-Vin; Chung, Joo-Ho; Hong, Seon-Pyo

    2005-09-07

    The genes were divided into seven categories according to biological function; apoptosis-related, immune response-related, signal transduction-related, cell cycle-related, cell growth-related, stress response-related and transcription-related genes. We compared the gene expression profiles of SNU-C4 cells between amygdalin-treated (5 mg/mL, 24 h) and non-treated groups using cDNA microarray analysis. We selected genes downregulated in cDNA microarray and investigated mRNA levels of the genes by RT-PCR. Microarray showed that amygdalin downregulated especially genes belonging to cell cycle category: exonuclease 1 (EXO1), ATP-binding cassette, sub-family F, member 2 (ABCF2), MRE11 meiotic recombination 11 homolog A (MRE11A), topoisomerase (DNA) I (TOP1), and FK506 binding protein 12-rapamycin-associated protein 1 (FRAP1). RT-PCR analysis revealed that mRNA levels of these genes were also decreased by amygdalin treatment in SNU-C4 human colon cancer cells. These results suggest that amygdalin have an anticancer effect via downregulation of cell cycle-related genes in SNU-C4 human colon cancer cells, and might be used for therapeutic anticancer drug.

  13. Advances in Gene/Cell Therapy in Epidermolysis Bullosa.

    Science.gov (United States)

    Murauer, Eva M; Koller, Ulrich; Pellegrini, Graziella; De Luca, Michele; Bauer, Johann W

    2015-01-01

    In the past few years, substantial preclinical and experimental advances have been made in the treatment of the severe monogenic skin blistering disease epidermolysis bullosa (EB). Promising approaches have been developed in the fields of protein and cell therapies, including allogeneic stem cell transplantation; in addition, the application of gene therapy approaches has become reality. The first ex vivo gene therapy for a junctional EB (JEB) patient was performed in Italy more than 8 years ago and was shown to be effective. We have now continued this approach for an Austrian JEB patient. Further, clinical trials for a gene therapy treatment of recessive dystrophic EB are currently under way in the United States and in Europe. In this review, we aim to point out that sustainable correction of autologous keratinocytes by stable genomic integration of a therapeutic gene represents a realistic option for patients with EB.

  14. Gene Silencing in Insect Cells Using RNAi.

    Science.gov (United States)

    Wu, Hsuan-Chen; March, John C; Bentley, William E

    2016-01-01

    A technique is described for synthesizing and transfecting double stranded RNA (dsRNA) for RNA interference (RNAi) in Sf-21 cell culture. Transfection with dsRNA only requires an hour and the cells usually recover within 12 h. Suggestions for designing dsRNA are included in the methods. Furthermore, websites are provided for rapid and effective dsRNA design. Three kits are essential for using the described methods: RNAqueous®-4PCR, Megascript™ T7 kit, and the Superscript™ III kit from Life Technologies, Inc.

  15. Involvement of distinct PKC gene products in T cell functions

    Directory of Open Access Journals (Sweden)

    Gottfried eBaier

    2012-08-01

    Full Text Available It is well established that members of the Protein kinase C(PKC family seem to have important roles in T cells. Focusing on the physiological and non-redundant PKC functions established in primary mouse T cells via germline gene-targeting approaches, our current knowledge defines two particularly critical PKC gene products, PKCθ and PKCα, as the flavor of PKC in T cells that appear to have a positive role in signaling pathways that are necessary for full antigen receptor-mediated T cell activation ex vivo and T cell-mediated immunity in vivo. Consistently, in spite of the current dogma that PKCθ inhibition might be sufficient to achieve complete immunosuppressive effects, more recent results have indicated that the pharmacological inhibition of PKCθ, and additionally, at least PKCα, appears to be needed to provide a successful approach for the prevention of allograft rejection and treatment of autoimmune diseases.

  16. Modeling of gene therapy for regenerative cells using intelligent agents.

    Science.gov (United States)

    Adly, Aya Sedky; Aboutabl, Amal Elsayed; Ibrahim, M Shaarawy

    2011-01-01

    Gene therapy is an exciting field that has attracted much interest since the first submission of clinical trials. Preliminary results were very encouraging and prompted many investigators and researchers. However, the ability of stem cells to differentiate into specific cell types holds immense potential for therapeutic use in gene therapy. Realization of this potential depends on efficient and optimized protocols for genetic manipulation of stem cells. It is widely recognized that gain/loss of function approaches using gene therapy are essential for understanding specific genes functions, and such approaches would be particularly valuable in studies involving stem cells. A significant complexity is that the development stage of vectors and their variety are still not sufficient to be efficiently applied in stem cell therapy. The development of scalable computer systems constitutes one step toward understanding dynamics of its potential. Therefore, the primary goal of this work is to develop a computer model that will support investigations of virus' behavior and organization on regenerative tissues including genetically modified stem cells. Different simulation scenarios were implemented, and their results were encouraging compared to ex vivo experiments, where the error rate lies in the range of acceptable values in this domain of application.

  17. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.

    Science.gov (United States)

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A F V

    2013-03-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

  18. Gene expression markers for Caenorhabditis elegans vulval cells.

    Science.gov (United States)

    Inoue, Takao; Sherwood, David R; Aspöck, Gudrun; Butler, James A; Gupta, Bhagwati P; Kirouac, Martha; Wang, Minqin; Lee, Pei-Yun; Kramer, James M; Hope, Ian; Bürglin, Thomas R; Sternberg, Paul W

    2002-12-01

    The analysis of cell fate patterning during the vulval development of Caenorhabditis elegans has relied mostly on the direct observation of cell divisions and cell movements (cell lineage analysis). However, reconstruction of the developing vulva from EM serial sections has suggested seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), many of which cannot be distinguished based on such observations. Here we report the vulval expression of seven genes, egl-17, cdh-3, ceh-2, zmp-1, B0034.1, T04B2.6 and F47B8.6 based on gfp, cfp and yfp (green fluorescent protein and color variants) reporter fusions. Each gene expresses in a specific subset of vulval cells, and is therefore useful as a marker for vulval cell fates. Together, expressions of markers distinguish six cell types, and reveal a strict temporal control of gene expression in the developing vulva.

  19. Impact of the cell division cycle on gene circuits

    Science.gov (United States)

    Bierbaum, Veronika; Klumpp, Stefan

    2015-12-01

    In growing cells, protein synthesis and cell growth are typically not synchronous, and, thus, protein concentrations vary over the cell division cycle. We have developed a theoretical description of genetic regulatory systems in bacteria that explicitly considers the cell division cycle to investigate its impact on gene expression. We calculate the cell-to-cell variations arising from cells being at different stages in the division cycle for unregulated genes and for basic regulatory mechanisms. These variations contribute to the extrinsic noise observed in single-cell experiments, and are most significant for proteins with short lifetimes. Negative autoregulation buffers against variation of protein concentration over the division cycle, but the effect is found to be relatively weak. Stronger buffering is achieved by an increased protein lifetime. Positive autoregulation can strongly amplify such variation if the parameters are set to values that lead to resonance-like behaviour. For cooperative positive autoregulation, the concentration variation over the division cycle diminishes the parameter region of bistability and modulates the switching times between the two stable states. The same effects are seen for a two-gene mutual-repression toggle switch. By contrast, an oscillatory circuit, the repressilator, is only weakly affected by the division cycle.

  20. Harnessing single cell sorting to identify cell division genes and regulators in bacteria.

    Directory of Open Access Journals (Sweden)

    Catherine Burke

    Full Text Available Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.

  1. Optimising gene therapy of hypoparathyroidism with hematopoietic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yi; L(U) Bing-jie; XU Ping; SONG Chun-fang

    2005-01-01

    Background The treatment of hypoparathyroidism (HPT) is still a difficult clinical problem, which necessitates a new therapy. Gene therapy of HPT has been valuable, but how to improve the gene transfer efficiency and expression stability is a problem. This study was designed to optimize the gene therapy of HPT with hematopoietic stem cells (HSCs) recombined with the parathyroid hormone (PTH) gene. Methods The human PTH gene was amplified by polymerase chain reaction (PCR) from pcDNA3.1-PTH vectors and inserted into murine stem cell virus (MSCV) vectors with double enzyme digestion (EcoRI and XhoI). The recombinant vectors were transfected into PA317 packaging cell lines by the lipofectin method and screened by G418 selective medium. The condensed recombinant retroviruses were extracted and used to infect HSCs, which were injected into mice suffering from HPT. The change of symptoms and serum levels of PTH and calcium in each group of mice were investigated. Results The human PTH gene was inserted into MSCV vectors successfully and the titres were up to 2×107 colony forming unit (CFU)/ml in condensed retroviral solution. The secretion of PTH reached 15 ng·10-6·cell-1 per 48 hours. The wild type viruses were not detected via PCR amplification, so they were safe for use. The mice suffering from HPT recovered quickly and the serum levels of calcium and PTH remained normal for about three months after the HSCs recombined with PTH were injected into them. The therapeutic effect of this method was better than simple recombinant retroviruses injection.Conclusions The recombinant retroviral vectors MSCV-PTH and the high-titre condensed retroviral solution recombined with the PTH gene are obtained. The recombinant retroviral solution could infect HSCs at a high rate of efficiency. The infected HSCs could cure HPT in mice. This method has provided theoretical evidence for the clinical gene therapy of HPT.

  2. Role of Hox genes in stem cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Anne Seifert; David F Werheid; Silvana M Knapp; Edda Tobiasch

    2015-01-01

    Hox genes are an evolutionary highly conserved genefamily. They determine the anterior-posterior body axisin bilateral organisms and influence the developmentalfate of cells. Embryonic stem cells are usually devoidof any Hox gene expression, but these transcriptionfactors are activated in varying spatial and temporalpatterns defining the development of various bodyregions. In the adult body, Hox genes are among othersresponsible for driving the differentiation of tissuestem cells towards their respective lineages in order torepair and maintain the correct function of tissues andorgans. Due to their involvement in the embryonic andadult body, they have been suggested to be useable forimproving stem cell differentiations in vitro and in vivo .In many studies Hox genes have been found as drivingfactors in stem cell differentiation towards adipogenesis,in lineages involved in bone and joint formation, mainlychondrogenesis and osteogenesis, in cardiovascularlineages including endothelial and smooth muscle celldifferentiations, and in neurogenesis. As life expectancyis rising, the demand for tissue reconstruction continuesto increase. Stem cells have become an increasinglypopular choice for creating therapies in regenerativemedicine due to their self-renewal and differentiationpotential. Especially mesenchymal stem cells are usedmore and more frequently due to their easy handlingand accessibility, combined with a low tumorgenicityand little ethical concerns. This review therefore intendsto summarize to date known correlations betweennatural Hox gene expression patterns in body tissuesand during the differentiation of various stem cellstowards their respective lineages with a major focus onmesenchymal stem cell differentiations. This overviewshall help to understand the complex interactions of Hoxgenes and differentiation processes all over the bodyas well as in vitro for further improvement of stem celltreatments in future regenerative medicine approaches.

  3. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    Science.gov (United States)

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

  4. Genome-editing Technologies for Gene and Cell Therapy

    Science.gov (United States)

    Maeder, Morgan L; Gersbach, Charles A

    2016-01-01

    Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed. PMID:26755333

  5. Genome-editing Technologies for Gene and Cell Therapy.

    Science.gov (United States)

    Maeder, Morgan L; Gersbach, Charles A

    2016-03-01

    Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed.

  6. Mural granulosa cell gene expression associated with oocyte developmental competence

    Directory of Open Access Journals (Sweden)

    Jiang Jin-Yi

    2010-03-01

    Full Text Available Abstract Background Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. Methods Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC. Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. Results The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox and nerve growth factor receptor associated protein 1 (Ngfrap1, which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2, which is involved in the regulation of extracellular matrix organization and biogenesis. Conclusions The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and

  7. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype.

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    Geert A Martens

    Full Text Available BACKGROUND AND METHODOLOGY: The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators. PRINCIPAL FINDINGS: A panel of 332 conserved beta cell biomarker genes was found to discriminate both isolated and laser capture microdissected beta cells from all other examined cell types. Of all conserved beta cell-markers, 15% were strongly beta cell-selective and functionally associated to hormone processing, 15% were shared with neuronal cells and associated to regulated synaptic vesicle transport and 30% with immune plus gut mucosal tissues reflecting active protein synthesis. Fasting specifically down-regulated the latter cluster, but preserved the neuronal and strongly beta cell-selective traits, indicating preserved differentiated state. Analysis of consensus binding site enrichment indicated major roles of CREB/ATF and various nutrient- or redox-regulated transcription factors in maintenance of differentiated beta cell phenotype. CONCLUSIONS: Conserved beta cell marker genes contain major gene clusters defined by their beta cell selectivity or by their additional abundance in either neural cells or in immune plus gut mucosal cells. This panel can be used as a template to identify changes in the differentiated state of beta cells.

  8. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  9. Gene Transfer in Eukaryotic Cells Using Activated Dendrimers

    Science.gov (United States)

    Dennig, Jörg

    Gene transfer into eukaryotic cells plays an important role in cell biology. Over the last 30 years a number of transfection methods have been developed to mediate gene transfer into eukaryotic cells. Classical methods include co-precipitation of DNA with calcium phosphate, charge-dependent precipitation of DNA with DEAE-dextran, electroporation of nucleic acids, and formation of transfection complexes between DNA and cationic liposomes. Gene transfer technologies based on activated PAMAM-dendrimers provide another class of transfection reagents. PAMAM-dendrimers are highly branched, spherical molecules. Activation of newly synthesized dendrimers involves hydrolytic removal of some of the branches, and results in a molecule with a higher degree of flexibility. Activated dendrimers assemble DNA into compact structures via charge interactions. Activated dendrimer - DNA complexes bind to the cell membrane of eukaryotic cells, and are transported into the cell by non-specific endocytosis. A structural model of the activated dendrimer - DNA complex and a potential mechanism for its uptake into cells will be discussed.

  10. Adenovirus gene transfer to amelogenesis imperfecta ameloblast-like cells.

    Directory of Open Access Journals (Sweden)

    Anton V Borovjagin

    Full Text Available To explore gene therapy strategies for amelogenesis imperfecta (AI, a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5 vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3 fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(vβ3/α(vβ5 integrins and heparan sulfate proteoglycans (HSPGs highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.

  11. A novel gene delivery system targeting cells expressing VEGF receptors

    Institute of Scientific and Technical Information of China (English)

    LIJUNMIN; JINGCHULUO; 等

    1999-01-01

    Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors.GV1,GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex.Using pSV2-β-galactosidase as a reporter gene,it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro.In vivo experiments,exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO,human malignant melanoma A375 and human hepatoma graft in nude mice.This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice.These results are correlated with the relevant receptors(flt-1,flk-1/KDR) expression on the targeted cells and tissues.

  12. Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells

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    Ana Paula Santin Bertoni

    2015-01-01

    Full Text Available Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times, P<0.0001; 2.39 times, P=0.01; 1.58 times, P=0.0003; and 1.87 times, P<0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P<0.0001; 1.75 times, P=0.037; and 1.95 times, P<0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P=0.069. These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.

  13. Blood cell gene expression profiling in rheumatoid arthritis. Discriminative genes and effect of rheumatoid factor

    DEFF Research Database (Denmark)

    Bovin, Lone Frier; Rieneck, Klaus; Workman, Christopher;

    2004-01-01

    To study the pathogenic importance of the rheumatoid factor (RF) in rheumatoid arthritis (RA) and to identify genes differentially expressed in patients and healthy individuals, total RNA was isolated from peripheral blood mononuclear cells (PBMC) from eight RF-positive and six RF-negative RA...... from all fourteen RA patients and healthy controls identified a subset of discriminative genes. These results were validated by real time reverse transcription polymerase chain reaction (RT-PCR) on another group of RA patients and healthy controls. This confirmed that the following genes had...

  14. Prediction of epigenetically regulated genes in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Loss, Leandro A; Sadanandam, Anguraj; Durinck, Steffen; Nautiyal, Shivani; Flaucher, Diane; Carlton, Victoria EH; Moorhead, Martin; Lu, Yontao; Gray, Joe W; Faham, Malek; Spellman, Paul; Parvin, Bahram

    2010-05-04

    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the

  15. Prediction of epigenetically regulated genes in breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Lu Yontao

    2010-06-01

    Full Text Available Abstract Background Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP. The pipeline (i reduces the dimensionality of the methylation data, (ii associates the reduced methylation data with gene expression data, and (iii ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i methylation sites are grouped across the genome to identify regions of interest, and (ii methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Results Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between

  16. New imaging probes to track cell fate: reporter genes in stem cell research.

    Science.gov (United States)

    Jurgielewicz, Piotr; Harmsen, Stefan; Wei, Elizabeth; Bachmann, Michael H; Ting, Richard; Aras, Omer

    2017-07-03

    Cell fate is a concept used to describe the differentiation and development of a cell in its organismal context over time. It is important in the field of regenerative medicine, where stem cell therapy holds much promise but is limited by our ability to assess its efficacy, which is mainly due to the inability to monitor what happens to the cells upon engraftment to the damaged tissue. Currently, several imaging modalities can be used to track cells in the clinical setting; however, they do not satisfy many of the criteria necessary to accurately assess several aspects of cell fate. In recent years, reporter genes have become a popular option for tracking transplanted cells, via various imaging modalities in small mammalian animal models. This review article examines the reporter gene strategies used in imaging modalities such as MRI, SPECT/PET, Optoacoustic and Bioluminescence Imaging. Strengths and limitations of the use of reporter genes in each modality are discussed.

  17. Gene transfection in primary stem-like cells of giant cell tumor of bone.

    Science.gov (United States)

    Singh, Shalini; Mak, Isabella; Power, Patricia; Cunningham, Melissa; Cunnigham, Melissa; Turcotte, Robert; Ghert, Michelle

    2010-01-01

    The neoplastic stem-like stromal cell of giant cell tumor of bone (GCT) survives for multiple passages in primary culture with a stable phenotype, and exhibits multipotent characteristics. The pathophysiology of this tumor has been studied through the primary culture of these cells. However, successful gene transfer of these cells has not been reported to date. In this short report, we describe the development of the first reported technique that results in efficient gene transfection in primary stem-like cells of GCT.

  18. Gene expression analysis of cell death induction by Taurolidine in different malignant cell lines

    Directory of Open Access Journals (Sweden)

    Weyhe Dirk

    2010-10-01

    Full Text Available Abstract Background The anti-infective agent Taurolidine (TRD has been shown to have cell death inducing properties, but the mechanism of its action is largely unknown. The aim of this study was to identify potential common target genes modulated at the transcriptional level following TRD treatment in tumour cell lines originating from different cancer types. Methods Five different malignant cell lines (HT29, Chang Liver, HT1080, AsPC-1 and BxPC-3 were incubated with TRD (100 μM, 250 μM and 1000 μM. Proliferation after 8 h and cell viability after 24 h were analyzed by BrdU assay and FACS analysis, respectively. Gene expression analyses were carried out using the Agilent -microarray platform to indentify genes which displayed conjoint regulation following the addition of TRD in all cell lines. Candidate genes were subjected to Ingenuity Pathways Analysis and selected genes were validated by qRT-PCR and Western Blot. Results TRD 250 μM caused a significant inhibition of proliferation as well as apoptotic cell death in all cell lines. Among cell death associated genes with the strongest regulation in gene expression, we identified pro-apoptotic transcription factors (EGR1, ATF3 as well as genes involved in the ER stress response (PPP1R15A, in ubiquitination (TRAF6 and mitochondrial apoptotic pathways (PMAIP1. Conclusions This is the first conjoint analysis of potential target genes of TRD which was performed simultaneously in different malignant cell lines. The results indicate that TRD might be involved in different signal transduction pathways leading to apoptosis.

  19. Gene expression analysis of in vivo fluorescent cells.

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    Konstantin Khodosevich

    Full Text Available BACKGROUND: The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD. Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters. METHODOLOGY/PRINCIPAL FINDINGS: We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of approximately 2ratio1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR. CONCLUSIONS/SIGNIFICANCE: Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.

  20. Identification of novel Notch target genes in T cell leukaemia

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    Warrander Fiona

    2009-06-01

    Full Text Available Abstract Background Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat. Results RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone or vectors containing constitutively active forms of Notch (N1ΔE or N3ΔE, and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1. Conclusion The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease.

  1. Gene expression during development of fetal and adult Leydig cells.

    Science.gov (United States)

    Dong, Lei; Jelinsky, Scott A; Finger, Joshua N; Johnston, Daniel S; Kopf, Gregory S; Sottas, Chantal M; Hardy, Matthew P; Ge, Ren-Shan

    2007-12-01

    In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.

  2. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells.

    Science.gov (United States)

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong

    2015-09-01

    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.

  3. Gene expression profiling and gene copy-number changes in malignant mesothelioma cell lines.

    Science.gov (United States)

    Zanazzi, Claudia; Hersmus, Remko; Veltman, Imke M; Gillis, Ad J M; van Drunen, Ellen; Beverloo, H Berna; Hegmans, Joost P J J; Verweij, Marielle; Lambrecht, Bart N; Oosterhuis, J Wolter; Looijenga, Leendert H J

    2007-10-01

    Malignant mesothelioma (MM) is an asbestos-induced tumor that acquires aneuploid DNA content during the tumorigenic process. We used instable MM cell lines as an in vitro model to study the impact of DNA copy-number changes on gene expression profiling, in the course of their chromosomal redistribution process. Two MM cell lines, PMR-MM2 (early passages of in vitro culture) and PMR-MM7 (both early and late passages of in vitro culture), were cytogenetically characterized. Genomic gains and losses were precisely defined using microarray-based comparative genomic hybridization (array-CGH), and minimal overlapping analysis led to the identification of the common unbalanced genomic regions. Using the U133Plus 2.0 Affymetrix gene chip array, we analyzed PMR-MM7 early and late passages for genome-wide gene expression, and correlated the differentially expressed genes with copy-number changes. The presence of a high number of genetic imbalances occurring from early to late culture steps reflected the tendency of MM cells toward genomic instability. The selection of specific chromosomal abnormalities observed during subsequent cultures demonstrated the spontaneous evolution of the cancer cells in an in vitro environment. MM cell lines were characterized by copy-number changes associated with the TP53 apoptotic pathway already present at the first steps of in vitro culture. Prolonged culture led to acquisition of additional chromosomal copy-number changes associated with dysregulation of genes involved in cell adhesion, regulation of mitotic cell cycle, signal transduction, carbohydrate metabolism, motor activity, glycosaminoglycan biosynthesis, protein binding activity, lipid transport, ATP synthesis, and methyltransferase activity.

  4. A Review of Gene Delivery and Stem Cell Based Therapies for Regenerating Inner Ear Hair Cells

    Directory of Open Access Journals (Sweden)

    Michael S. Detamore

    2011-09-01

    Full Text Available Sensory neural hearing loss and vestibular dysfunction have become the most common forms of sensory defects, affecting millions of people worldwide. Developing effective therapies to restore hearing loss is challenging, owing to the limited regenerative capacity of the inner ear hair cells. With recent advances in understanding the developmental biology of mammalian and non-mammalian hair cells a variety of strategies have emerged to restore lost hair cells are being developed. Two predominant strategies have developed to restore hair cells: transfer of genes responsible for hair cell genesis and replacement of missing cells via transfer of stem cells. In this review article, we evaluate the use of several genes involved in hair cell regeneration, the advantages and disadvantages of the different viral vectors employed in inner ear gene delivery and the insights gained from the use of embryonic, adult and induced pluripotent stem cells in generating inner ear hair cells. Understanding the role of genes, vectors and stem cells in therapeutic strategies led us to explore potential solutions to overcome the limitations associated with their use in hair cell regeneration.

  5. Gene expression analysis of dendritic/Langerhans cells and Langerhans cell histiocytosis

    NARCIS (Netherlands)

    Rust, Renata; Kluiver, J.; Visser, Lydia; Harms, G.; Blokzijl, T.; Kamps, W.A.; Poppema, Sibrand; van den Berg, Anke

    2006-01-01

    Langerhans cell histiocytosis (LCH) is a neoplastic disorder that results in clonal proliferation of cells with a Langerhans cell (LQ phenotype. The pathogenesis of LCH is still poorly understood. In the present study, serial analysis of gene expression (SAGE) was applied to LCs generated from umbil

  6. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...

  7. The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Zandi, Roza; Xu, Kai; Poulsen, Hans S

    2011-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone...... or in combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth...

  8. Detection of gene expression in an individual cell type within a cell mixture using microarray analysis.

    Directory of Open Access Journals (Sweden)

    Penelope A Bryant

    Full Text Available BACKGROUND: A central issue in the design of microarray-based analysis of global gene expression is the choice between using cells of single type and a mixture of cells. This study quantified the proportion of lipopolysaccharide (LPS induced differentially expressed monocyte genes that could be measured in peripheral blood mononuclear cells (PBMC, and determined the extent to which gene expression in the non-monocyte cell fraction diluted or obscured fold changes that could be detected in the cell mixture. METHODOLOGY/PRINCIPAL FINDINGS: Human PBMC were stimulated with LPS, and monocytes were then isolated by positive (Mono+ or negative (Mono- selection. The non-monocyte cell fraction (MonoD remaining after positive selection of monocytes was used to determine the effect of non-monocyte cells on overall expression. RNA from LPS-stimulated PBMC, Mono+, Mono- and MonoD samples was co-hybridised with unstimulated RNA for each cell type on oligonucleotide microarrays. There was a positive correlation in gene expression between PBMC and both Mono+ (0.77 and Mono- (0.61-0.67 samples. Analysis of individual genes that were differentially expressed in Mono+ and Mono- samples showed that the ability to detect expression of some genes was similar when analysing PBMC, but for others, differential expression was either not detected or changed in the opposite direction. As a result of the dilutional or obscuring effect of gene expression in non-monocyte cells, overall about half of the statistically significant LPS-induced changes in gene expression in monocytes were not detected in PBMC. However, 97% of genes with a four fold or greater change in expression in monocytes after LPS stimulation, and almost all (96-100% of the top 100 most differentially expressed monocyte genes were detected in PBMC. CONCLUSIONS/SIGNIFICANCE: The effect of non-responding cells in a mixture dilutes or obscures the detection of subtle changes in gene expression in an individual

  9. The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells

    DEFF Research Database (Denmark)

    Zandi, Roza; Xu, Kai; Poulsen, Hans S

    2011-01-01

    The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone...... or in combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth...

  10. Identification of the T-cell receptor alpha variable (TRAV) gene(s) in T-cell malignancies.

    Science.gov (United States)

    Hinz, T; Kabelitz, D

    2000-12-01

    Due to the lack of a complete range of monoclonal antibodies (mAb) it is often impossible to rapidly identify by flow cytometry the T-cell receptor variable genes in patients suffering from T-cell malignancies. This applies especially to the alpha variable genes (TRAV), since only very few anti-TcR variable alpha mAb are available. We describe a very rapid method for inverse PCR amplification of the TcR alpha chain without prior purification of the double-stranded cDNA, provide the sequences for appropriate oligonucleotides, and describe a buffer system that dramatically enhances the amplification efficiency as compared to standard conditions.

  11. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  12. Toward a stem cell gene therapy for breast cancer.

    Science.gov (United States)

    Li, ZongYi; Liu, Ying; Tuve, Sebastian; Xun, Ye; Fan, Xiaolong; Min, Liang; Feng, Qinghua; Kiviat, Nancy; Kiem, Hans-Peter; Disis, Mary Leonora; Lieber, André

    2009-05-28

    Current approaches for treatment of late-stage breast cancer rarely result in a long-term cure. In part this is due to tumor stroma that prevents access of systemically or intratumorally applied therapeutics. We propose a stem cell gene therapy approach for controlled tumor stroma degradation that uses the pathophysiologic process of recruitment of inflammatory cells into the tumor. This approach involves genetic modification of hematopoietic stem cells (HSCs) and their subsequent transplantation into tumor-bearing mice. We show that inducible, intratumoral expression of relaxin (Rlx) either by transplanting tumor cells that contained the Rlx gene or by transplantation of mouse HSCs transduced with an Rlx-expressing lentivirus vector delays tumor growth in a mouse model of breast cancer. The antitumor effect of Rlx was mediated through degradation of tumor stroma, which provided increased access of infiltrating antitumor immune cells to their target tumor cells. Furthermore, we have shown in a human/mouse chimeric model that genetically modified HSCs expressing a transgene can access the tumor site. Our findings are relevant for cancer gene therapy and immunotherapy.

  13. Direct Cell Lysis for Single-Cell Gene Expression Profiling

    OpenAIRE

    David eSvec; Daniel eAndersson; Milos ePekny; Robert eSjöback; Mikael eKubista; Anders eStåhlberg

    2013-01-01

    The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously express...

  14. Double suicide genes selectively kill human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Liu Lunxu

    2011-02-01

    Full Text Available Abstract Background To construct a recombinant adenovirus containing CDglyTK double suicide genes and evaluate the killing effect of the double suicide genes driven by kinase domain insert containing receptor (KDR promoter on human umbilical vein endothelial cells. Methods Human KDR promoter, Escherichia coli (E. coli cytosine deaminase (CD gene and the herpes simplex virus-thymidine kinase (TK gene were cloned using polymerase chain reaction (PCR. Plasmid pKDR-CDglyTK was constructed with the KDR promoter and CDglyTK genes. A recombinant adenoviral plasmid AdKDR-CDglyTK was then constructed and transfected into 293 packaging cells to grow and harvest adenoviruses. KDR-expressing human umbilical vein endothelial cells (ECV304 and KDR-negative liver cancer cell line (HepG2 were infected with the recombinant adenoviruses at different multiplicity of infection (MOI. The infection rate was measured by green fluorescent protein (GFP expression. The infected cells were cultured in culture media containing different concentrations of prodrugs ganciclovir (GCV and/or 5-fluorocytosine (5-FC. The killing effects were measured using two different methods, i.e. annexin V-FITC staining and terminal transferase-mediated dUTP nick end-labeling (TUNEL staining. Results Recombinant adenoviruses AdKDR-CDglyTK were successfully constructed and they infected ECV304 and HepG2 cells efficiently. The infection rate was dependent on MOI of recombinant adenoviruses. ECV304 cells infected with AdKDR-CDglyTK were highly sensitive to GCV and 5-FC. The cell survival rate was dependent on both the concentration of the prodrugs and the MOI of recombinant adenoviruses. In contrast, there were no killing effects in the HepG2 cells. The combination of two prodrugs was much more effective in killing ECV304 cells than GCV or 5-FC alone. The growth of transgenic ECV304 cells was suppressed in the presence of prodrugs. Conclusion AdKDR-CDglyTK/double prodrog system may be a useful

  15. Specifically targeted gene therapy for small-cell lung cancer

    DEFF Research Database (Denmark)

    Christensen, C.L.; Zandi, R.; Gjetting, T.

    2009-01-01

    Small-cell lung cancer (SCLC) is a highly malignant disease with poor prognosis. Hence, there is great demand for new therapies that can replace or supplement the current available treatment regimes. Gene therapy constitutes a promising strategy and relies on the principle of introducing exogenous....... This review describes and discusses the current status of the application of gene therapy in relation to SCLC Udgivelsesdato: 2009/4...... DNA into malignant cells causing them to die. Since SCLC is a highly disseminated malignancy, the gene therapeutic agent must be administered systemically, obligating a high level of targeting of tumor tissue and the use of delivery vehicles designed for systemic circulation of the therapeutic DNA...

  16. Lentiviral hematopoietic stem cell gene therapy in inherited metabolic disorders

    NARCIS (Netherlands)

    G. Wagemaker (Gerard)

    2014-01-01

    textabstractAfter more than 20 years of development, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and storage disorders. This brief review summarizes the development and applications, focusing on the lysosomal enzyme

  17. Adenovirus as a gene therapy vector for hematopoietic cells.

    Science.gov (United States)

    Marini, F C; Yu, Q; Wickham, T; Kovesdi, I; Andreeff, M

    2000-06-01

    Adenovirus (Adv)-mediated gene transfer has recently gained new attention as a means to deliver genes for hematopoietic stem cell (HSC) or progenitor cell gene therapy. In the past, HSCs have been regarded as poor Adv targets, mainly because they lack the specific Adv receptors required for efficient and productive Adv infection. In addition, the nonintegrating nature of Adv has prevented its application to HSC and bone marrow transduction protocols where long-term expression is required. There is even controversy as to whether Adv can infect hematopoietic cells at all. In fact, the ability of Adv to infect epithelium-based targets and its inability to effectively transfect HSCs have been used in the development of eradication schemes that use Adv to preferentially infect and "purge" tumor cell-contaminating HSC grafts. However, there are data supporting the existence of productive Adv infections into HSCs. Such protocols involve the application of cytokine mixtures, high multiplicities of infection, long incubation periods, and more recently, immunological and genetic modifications to Adv itself to enable it to efficiently transfer genes into HSCs. This is a rapidly growing field, both in terms of techniques and applications. This review examines the two sides of the Adv/CD34 controversy as well as the current developments in this field.

  18. Gene targeting in embryonic stem cells, II: conditional technologies

    Science.gov (United States)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  19. Similarity in gene-regulatory networks suggests that cancer cells share characteristics of embryonic neural cells.

    Science.gov (United States)

    Zhang, Zan; Lei, Anhua; Xu, Liyang; Chen, Lu; Chen, Yonglong; Zhang, Xuena; Gao, Yan; Yang, Xiaoli; Zhang, Min; Cao, Ying

    2017-08-04

    Cancer cells are immature cells resulting from cellular reprogramming by gene misregulation, and redifferentiation is expected to reduce malignancy. It is unclear, however, whether cancer cells can undergo terminal differentiation. Here, we show that inhibition of the epigenetic modification enzyme enhancer of zeste homolog 2 (EZH2), histone deacetylases 1 and 3 (HDAC1 and -3), lysine demethylase 1A (LSD1), or DNA methyltransferase 1 (DNMT1), which all promote cancer development and progression, leads to postmitotic neuron-like differentiation with loss of malignant features in distinct solid cancer cell lines. The regulatory effect of these enzymes in neuronal differentiation resided in their intrinsic activity in embryonic neural precursor/progenitor cells. We further found that a major part of pan-cancer-promoting genes and the signal transducers of the pan-cancer-promoting signaling pathways, including the epithelial-to-mesenchymal transition (EMT) mesenchymal marker genes, display neural specific expression during embryonic neurulation. In contrast, many tumor suppressor genes, including the EMT epithelial marker gene that encodes cadherin 1 (CDH1), exhibited non-neural or no expression. This correlation indicated that cancer cells and embryonic neural cells share a regulatory network, mediating both tumorigenesis and neural development. This observed similarity in regulatory mechanisms suggests that cancer cells might share characteristics of embryonic neural cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells

    OpenAIRE

    Holkers, M.; Maggio, I.; Liu, J.; Janssen, J.M.; Miselli, F; Mussolino, C.; Recchia, A; Cathomen, T.; Goncalves, M. A. F. V.

    2012-01-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehi...

  1. [Effect of Pinch-3 gene interference of glomerular podocytes on cell morphology and cell traction force].

    Science.gov (United States)

    Yang, Yu; Niu, Qingyuan; Ji, Zhenling; Zhang, Jingjing; Li, Jianting; Ma, Deshun

    2013-06-01

    Pinch-3 protein is an important constituent of cell membranes, which directly affects the cell morphology and mechanical properties. We observed and compared the change of morphology and cell traction force of glomerular podocytes before and after Pinch-3 gene inhibition by gene interference technology in this experiment. We found that a number of pores appeared on the cell surface, and the cell projected area were increased at the same time, with an approximate average about an increase of 40% after Pinch-3 gene inhibition. The results showed that the cell traction force of glomerular podocytes was significantly reduced, with an approximate average decrease of 40%, the maximum value of the cell traction force was reduced and the distribution of cell traction force became dispersive. All this suggested that after Pinch-3 gene inhibition, some pores created on the cell surface influenced the physical properties of glomerular podocytes and then affected the cell projected area and influenced the formation and distribution of cell traction force of the glomerular podocytes as well.

  2. Differentially expressed genes in giant cell tumor of bone.

    Science.gov (United States)

    Babeto, Erica; Conceição, André Luis Giacometti; Valsechi, Marina Curado; Peitl Junior, Paulo; de Campos Zuccari, Débora Aparecida Pires; de Lima, Luiz Guilherme Cernaglia Aureliano; Bonilha, Jane Lopes; de Freitas Calmon, Marília; Cordeiro, José Antônio; Rahal, Paula

    2011-04-01

    Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.

  3. RNA Interference Targeting Leptin Gene Effect on Hepatic Stellate Cells

    Institute of Scientific and Technical Information of China (English)

    XUE Xiulan; LIN Jusheng; SONG Yuhu; SUN Xuemei; ZHOU Hejun

    2005-01-01

    To construct the specific siRNA expression vectors and investigate their effect on leptin and collagen I in HSC, which provide a new approach to the prevent and treat hepatic fibrosis. The five siRNAs against leptin gene were transcript synthesized intracellularly by expression templates of plasmid vector psiRNA-hH1neo. The recombinant leptin siRNA plasmid vectors could express in eukaryocyte , and then to evaluate them by using enzyme cutting and sequencing. The recombinant plasmids were transfected into HSCs using Lipofectamine methods respectively. The cells were selected after growing in DMEM containing 300 μg/mL G418 for about 4 weeks. Gene expression of leptin and collagen I were showed by Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Identification by enzyme cutting and sequencing showed that the leptin siRNA expression vectors were constructed successfully, and leptin siRNA could inhibit the leptin and collagen I gene expression effectively. It was concluded that RNA interference-mediated silencing of leptin gene diminished leptin and collagen I gene expression in HSCs. Furthermore, attenuated the extracellular matrix over-deposition at the same time. Leptin gene is ideal targets of gene therapy for liver fibrosis.

  4. A survey of disease connections for CD4+ T cell master genes and their directly linked genes.

    Science.gov (United States)

    Li, Wentian; Espinal-Enríquez, Jesús; Simpfendorfer, Kim R; Hernández-Lemus, Enrique

    2015-12-01

    Genome-wide association studies and other genetic analyses have identified a large number of genes and variants implicating a variety of disease etiological mechanisms. It is imperative for the study of human diseases to put these genetic findings into a coherent functional context. Here we use system biology tools to examine disease connections of five master genes for CD4+ T cell subtypes (TBX21, GATA3, RORC, BCL6, and FOXP3). We compiled a list of genes functionally interacting (protein-protein interaction, or by acting in the same pathway) with the master genes, then we surveyed the disease connections, either by experimental evidence or by genetic association. Embryonic lethal genes (also known as essential genes) are over-represented in master genes and their interacting genes (55% versus 40% in other genes). Transcription factors are significantly enriched among genes interacting with the master genes (63% versus 10% in other genes). Predicted haploinsufficiency is a feature of most these genes. Disease-connected genes are enriched in this list of genes: 42% of these genes have a disease connection according to Online Mendelian Inheritance in Man (OMIM) (versus 23% in other genes), and 74% are associated with some diseases or phenotype in a Genome Wide Association Study (GWAS) (versus 43% in other genes). Seemingly, not all of the diseases connected to genes surveyed were immune related, which may indicate pleiotropic functions of the master regulator genes and associated genes.

  5. Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

    Institute of Scientific and Technical Information of China (English)

    Claudia Lange; Philipp Bassler; Michael V. Lioznov; Helge Bruns; Dietrich Kluth; Axel R. Zander; Henning C. Fiegel

    2005-01-01

    AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore,we assessed the influence of cocultured liver cells on induction of liver-specific gene expression.METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF,EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19,and AFP was performed in the different cell populations.RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18,CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression.CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.

  6. Blood cell gene expression profiling in rheumatoid arthritis. Discriminative genes and effect of rheumatoid factor

    DEFF Research Database (Denmark)

    Bovin, Lone Frier; Rieneck, Klaus; Workman, Christopher;

    2004-01-01

    To study the pathogenic importance of the rheumatoid factor (RF) in rheumatoid arthritis (RA) and to identify genes differentially expressed in patients and healthy individuals, total RNA was isolated from peripheral blood mononuclear cells (PBMC) from eight RF-positive and six RF-negative RA...

  7. Blood cell gene expression profiling in rheumatoid arthritis - Discriminative genes and effect of rheumatoid factor

    DEFF Research Database (Denmark)

    Bovin, L.F.; Rieneck, K.; Workman, Christopher;

    2004-01-01

    To study the pathogenic importance of the rheumatoid factor (RF) in rheumatoid arthritis (RA) and to identify genes differentially expressed in patients and healthy individuals, total RNA was isolated from peripheral blood mononuclear cells (PBMC) from eight RF-positive and six RF-negative RA...

  8. Profiling helper T cell subset gene expression in deer mice

    Directory of Open Access Journals (Sweden)

    Hjelle Brian

    2006-08-01

    Full Text Available Abstract Background Deer mice (Peromyscus maniculatus are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV, the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT, Th2 cells (GATA-3, STAT6, IL-4, IL-5 and regulatory T cells (Fox-p3, IL-10, TGFβ1. These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

  9. [Meta-analysis of oral squamous cell carcinoma on gene expression level].

    Science.gov (United States)

    Shao, Yang; Liang, Yan; Leng, Dong

    2014-01-01

    To study the differently expressed genes of oral squamous cell carcinoma (OSCC) tissue. Gene expression datasets related to oral squamous cell carcinoma in the gene expression omnibus (gene expression omnibus, GEO) repository were retrieved. Datasets were merged by normalization.Significantly expressed genes were obtained by statistical methods, and genes' functions, interactions, signaling pathways were analyzed accordingly. In GEO, there were 1 125 records related to OSCC, and four of them were selected and merged to a super array data, within the super array data, 233 genes were significantly expressed (P expressed genes were selected as signature genes.Signature genes were more related to cell surface or cell-cell interactive activities. Clusters of interactive signature genes and the related signaling pathways were related with mitosis process. OSCC signature genes and the corresponding signaling pathways will provide not only an important clue for further research of the disease, but also reference for diagnosis and treatment.

  10. [Selection of retroviral vector producing cell lines and gene transfer into hematopoietic cells].

    Science.gov (United States)

    Bagnis, C; Mannoni, P

    1996-04-01

    Transduction and expression of a transgene in hematopoietic stem cells with retroviral vectors still remain major challenges for gene therapy in blood disorders. Use of an easily detectable gene marker, such as the nlsLacZ, at the laboratory and clinical levels, provides a powerful approach of these two combined problems.

  11. WT1-specific T cell receptor gene therapy: improving TCR function in transduced T cells.

    Science.gov (United States)

    Stauss, Hans J; Thomas, Sharyn; Cesco-Gaspere, Michela; Hart, Daniel P; Xue, Shao-An; Holler, Angelika; King, Judy; Wright, Graham; Perro, Mario; Pospori, Constantina; Morris, Emma

    2008-01-01

    Adoptive transfer of antigen-specific T lymphocytes is an attractive form of immunotherapy for haematological malignancies and cancer. The difficulty of isolating antigen-specific T lymphocytes for individual patients limits the more widespread use of adoptive T cell therapy. The demonstration that cloned T cell receptor (TCR) genes can be used to produce T lymphocyte populations of desired specificity offers new opportunities for antigen-specific T cell therapy. The first trial in humans demonstrated that TCR gene-modified T cells persisted for an extended time period and reduced tumor burden in some patients. The WT1 protein is an attractive target for immunotherapy of leukemia and solid cancer since elevated expression has been demonstrated in AML, CML, MDS and in breast, colon and ovarian cancer. In the past, we have isolated high avidity CTL specific for a WT1-derived peptide presented by HLA-A2 and cloned the TCR alpha and beta genes of a WT1-specific CTL line. The genes were inserted into retroviral vectors for transduction of human peripheral blood T lymphocytes of leukemia patients and normal donors. The treatment of leukemia-bearing NOD/SCID mice with T cells transduced with the WT1-specific TCR eliminated leukemia cells in the bone marrow of most mice, while treatment with T cells transduced with a TCR of irrelevant specificity did not diminish the leukemia burden. In order to improve the safety and efficacy of TCR gene therapy, we have developed lentiviral TCR gene transfer. In addition, we employed strategies to enhance TCR expression while avoiding TCR mis-pairing. It may be possible to generate dominant TCR constructs that can suppress the expression of the endogenous TCR on the surface of transduced T cells. The development of new TCR gene constructs holds great promise for the safe and effective delivery of TCR gene therapy for the treatment of malignancies.

  12. Identifying genes that mediate anthracyline toxicity in immune cells

    Directory of Open Access Journals (Sweden)

    Amber eFrick

    2015-04-01

    Full Text Available The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS, we identified four genome-wide significant quantitative trait loci (QTL that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01x10-8. Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05.In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Thus, further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies.

  13. Expression QTL mapping in regulatory and helper T cells from the BXD family of strains reveals novel cell-specific genes, gene-gene interactions and candidate genes for auto-immune disease

    Directory of Open Access Journals (Sweden)

    Alberts Rudi

    2011-12-01

    Full Text Available Abstract Background Regulatory T cells (Tregs play an essential role in the control of the immune response. Treg cells represent important targets for therapeutic interventions of the immune system. Therefore, it will be very important to understand in more detail which genes are specifically activated in Treg cells versus T helper (Th cells, and which gene regulatory circuits may be involved in specifying and maintaining Treg cell homeostasis. Results We isolated Treg and Th cells from a genetically diverse family of 31 BXD type recombinant inbred strains and the fully inbred parental strains of this family--C57BL/6J and DBA/2J. Subsequently genome-wide gene expression studies were performed from the isolated Treg and Th cells. A comparative analysis of the transcriptomes of these cell populations allowed us to identify many novel differentially expressed genes. Analysis of cis- and trans-expression Quantitative Trait Loci (eQTLs highlighted common and unique regulatory mechanisms that are active in the two cell types. Trans-eQTL regions were found for the Treg functional genes Nrp1, Stat3 and Ikzf4. Analyses of the respective QTL intervals suggested several candidate genes that may be involved in regulating these genes in Treg cells. Similarly, possible candidate genes were found which may regulate the expression of F2rl1, Ctla4, Klrb1f. In addition, we identified a focused group of candidate genes that may be important for the maintenance of self-tolerance and the prevention of allergy. Conclusions Variation of expression across the strains allowed us to find many novel gene-interaction networks in both T cell subsets. In addition, these two data sets enabled us to identify many differentially expressed genes and to nominate candidate genes that may have important functions for the maintenance of self-tolerance and the prevention of allergy.

  14. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis

    Science.gov (United States)

    Størling, Joachim; Pociot, Flemming

    2017-01-01

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis. PMID:28212332

  15. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis.

    Science.gov (United States)

    Størling, Joachim; Pociot, Flemming

    2017-02-16

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis.

  16. T-cell suicide gene therapy prompts thymic renewal in adults after hematopoietic stem cell transplantation.

    Science.gov (United States)

    Vago, Luca; Oliveira, Giacomo; Bondanza, Attilio; Noviello, Maddalena; Soldati, Corrado; Ghio, Domenico; Brigida, Immacolata; Greco, Raffaella; Lupo Stanghellini, Maria Teresa; Peccatori, Jacopo; Fracchia, Sergio; Del Fiacco, Matteo; Traversari, Catia; Aiuti, Alessandro; Del Maschio, Alessandro; Bordignon, Claudio; Ciceri, Fabio; Bonini, Chiara

    2012-08-30

    The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+ -cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31+ recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution.

  17. Planar cell polarity genes and neural tube closure.

    Science.gov (United States)

    Ueno, Naoto; Greene, Nicholas D E

    2003-11-01

    Closure of the neural tube is essential for normal development of the brain and spinal cord. Failure of closure results in neural tube defects (NTDs), common and clinically severe congenital malformations whose molecular mechanisms remain poorly understood. On the other hand, it is increasingly well established that common molecular mechanisms are employed to regulate morphogenesis of multicellular organisms. For example, signaling triggered by polypeptide growth factors is highly conserved among species and utilized in multiple developmental processes. Recent studies have revealed that the Drosophila planar cell polarity (PCP) pathway, which directs position and direction of wing hairs on the surface of the fly wing, is well conserved, and orthologs of several genes encoding components of the pathway are also found in vertebrates. Interestingly, in vertebrates, this signaling pathway appears to be co-opted to regulate "convergent extension" cell movements during gastrulation. Disruption of vertebrate PCP genes in Xenopus laevis or zebrafish causes severe gastrulation defects or the shortening of the trunk, as well as mediolateral expansion of somites. In Xenopus, in which the neural tube closes by elevation and fusion of neural folds, inhibition of convergent extension can also prevent neural tube closure causing a "spina bifida-like" appearance. Furthermore, several of the genes involved in the PCP pathway have recently been shown to be required for neural tube closure in the mouse, since mutation of these genes causes NTDs. Therefore, understanding the mechanisms underlying the establishment of cell polarity in Drosophila may provide important clues to the molecular basis of NTDs.

  18. Early gene regulation of osteogenesis in embryonic stem cells

    KAUST Repository

    Kirkham, Glen R.

    2012-01-01

    The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFβ1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data. © 2012 The Royal Society of Chemistry.

  19. Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Zhang Ping

    2006-09-01

    Full Text Available Abstract Background Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells. Methods A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated. Results Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells. Conclusion The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis

  20. Recombinant cells that highly express chromosomally-integrated heterologous gene

    Science.gov (United States)

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  1. Nitric oxide and oxygen radicals induced apoptosis via bcl-2 and p53 pathway in hypoxia-reoxygenated cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    SHEN; Jiangang; (沈剑刚); QIU; Xingshen; (丘幸生); JIANG; Bo; (姜; 泊); ZHANG; Deliang; (张德良); XIN; Wenjuan; (忻文娟); Peter; C.W.; Fung; ZHAO; Baolu; (赵保路)

    2003-01-01

    Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygenation further increased both apoptotic and necrotic cell death. NO, TBARS, DNA fragmentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, and inhibited DNA fragmentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 pathway in hypoxic and hypoxia-reoxygenated cardiomyocytes.

  2. T cells and gene regulation: the switching on and turning up of genes after T cell receptor stimulation in CD8 T cells

    Directory of Open Access Journals (Sweden)

    James M Conley

    2016-02-01

    Full Text Available Signaling downstream of the T cell receptor (TCR is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength. T cells rapidly alter their gene expression upon activation. Multiple signaling pathways downstream of the TCR activate transcription factors, which are critical for this process. The dynamics between proximal TCR signaling, transcription factor activation, and CD8 T cell function are discussed here. We propose that Inducible T cell kinase (ITK acts as a rheostat for gene expression. This unique regulation of TCR signaling by ITK provides a possible signaling mechanism for the promotion of a diverse T cell repertoire in response to pathogen.

  3. T Cells and Gene Regulation: The Switching On and Turning Up of Genes after T Cell Receptor Stimulation in CD8 T Cells

    Science.gov (United States)

    Conley, James M.; Gallagher, Michael P.; Berg, Leslie J.

    2016-01-01

    Signaling downstream of the T cell receptor (TCR) is directly regulated by the dose and affinity of peptide antigen. The strength of TCR signaling drives a multitude of T cell functions from development to differentiation. CD8 T cells differentiate into a diverse pool of effector and memory cells after activation, a process that is critical for pathogen clearance and is highly regulated by TCR signal strength. T cells rapidly alter their gene expression upon activation. Multiple signaling pathways downstream of the TCR activate transcription factors, which are critical for this process. The dynamics between proximal TCR signaling, transcription factor activation and CD8 T cell function are discussed here. We propose that inducible T cell kinase (ITK) acts as a rheostat for gene expression. This unique regulation of TCR signaling by ITK provides a possible signaling mechanism for the promotion of a diverse T cell repertoire in response to pathogen. PMID:26973653

  4. Effect of promoter architecture on the cell-to-cell variability in gene expression.

    Directory of Open Access Journals (Sweden)

    Alvaro Sanchez

    2011-03-01

    Full Text Available According to recent experimental evidence, promoter architecture, defined by the number, strength and regulatory role of the operators that control transcription, plays a major role in determining the level of cell-to-cell variability in gene expression. These quantitative experiments call for a corresponding modeling effort that addresses the question of how changes in promoter architecture affect variability in gene expression in a systematic rather than case-by-case fashion. In this article we make such a systematic investigation, based on a microscopic model of gene regulation that incorporates stochastic effects. In particular, we show how operator strength and operator multiplicity affect this variability. We examine different modes of transcription factor binding to complex promoters (cooperative, independent, simultaneous and how each of these affects the level of variability in transcriptional output from cell-to-cell. We propose that direct comparison between in vivo single-cell experiments and theoretical predictions for the moments of the probability distribution of mRNA number per cell can be used to test kinetic models of gene regulation. The emphasis of the discussion is on prokaryotic gene regulation, but our analysis can be extended to eukaryotic cells as well.

  5. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture......The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...

  6. Gene expression profile differences in high and low metastatic human ovarian cancer cell lines by gene chip

    Institute of Scientific and Technical Information of China (English)

    许沈华; 牟瀚舟; 吕桂泉; 朱赤红; 羊正炎; 高永良; 楼洪坤; 刘祥麟; 程勇; 杨文

    2002-01-01

    Objectives To study the difference between gene expressions of high (H0-8910PM) and low (HO-8910) metastatic human ovarian carcinoma cell lines and screen novel associated genes by cDNA microarray. Methods cDNA retro-transcribed from equal quantities of mRNA derived from high and low metastatic tumor cells or normal ovarian tissues were labeled with Cy5 and Cy3 fluorescein as probes. The mixed probe was hybridized with two pieces of BioDoor 4096 double dot human whole gene chip and scanned with a ScanArray 3000 laser scanner. The acquired image was analyzed by ImaGene 3.0 software. Results A total of 355 genes with expression levels more than 3 times larger were found by comparing the HO-8910 cell with normal ovarian epithelial cells. A total of 323 genes with expression levels more than 3 times larger in HO-8910PM cells compared to normal ovarian epithelium cells were also detected. A total of 165 genes whose expression levels were more than two times those of HO-8910PM cells compared to their mother cell line (HO-8910) were detected. Twenty-one genes with expression levels >3 times were found from comparison of these two tumor cell lines.Conclusions cDNA microarray techniques are effective in screening differential gene expression between two human ovarian cancer cell lines (H0-8910PM; HO-8910) and normal ovarian epithelial cells. These genes may be related to the genesis and development of ovarian carcinoma. Analysis of the human ovarian cancer gene expression profile with cDNA microarray may help in gene diagnosis, treatment and prevention.

  7. Comparative Gene Expression Profiling of Primary and Metastatic Renal Cell Carcinoma Stem Cell-Like Cancer Cells.

    Science.gov (United States)

    Khan, Mohammed I; Czarnecka, Anna M; Lewicki, Sławomir; Helbrecht, Igor; Brodaczewska, Klaudia; Koch, Irena; Zdanowski, Robert; Król, Magdalena; Szczylik, Cezary

    2016-01-01

    Recent advancement in cancer research has shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations are found in a single tumor. Cancer development and tumor growth are driven by specific types of cells-stem cell-like cancer cells (SCLCCs)-which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markers-CD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilent's human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have

  8. Discrimination of meniscal cell phenotypes using gene expression profiles

    Directory of Open Access Journals (Sweden)

    M Son

    2012-03-01

    Full Text Available The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus. The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

  9. The FRIABLE1 gene product affects cell adhesion in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Lutz Neumetzler

    Full Text Available Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1, was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246. Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.

  10. Embryo quality predictive models based on cumulus cells gene expression

    Directory of Open Access Journals (Sweden)

    Devjak R

    2016-06-01

    Full Text Available Since the introduction of in vitro fertilization (IVF in clinical practice of infertility treatment, the indicators for high quality embryos were investigated. Cumulus cells (CC have a specific gene expression profile according to the developmental potential of the oocyte they are surrounding, and therefore, specific gene expression could be used as a biomarker. The aim of our study was to combine more than one biomarker to observe improvement in prediction value of embryo development. In this study, 58 CC samples from 17 IVF patients were analyzed. This study was approved by the Republic of Slovenia National Medical Ethics Committee. Gene expression analysis [quantitative real time polymerase chain reaction (qPCR] for five genes, analyzed according to embryo quality level, was performed. Two prediction models were tested for embryo quality prediction: a binary logistic and a decision tree model. As the main outcome, gene expression levels for five genes were taken and the area under the curve (AUC for two prediction models were calculated. Among tested genes, AMHR2 and LIF showed significant expression difference between high quality and low quality embryos. These two genes were used for the construction of two prediction models: the binary logistic model yielded an AUC of 0.72 ± 0.08 and the decision tree model yielded an AUC of 0.73 ± 0.03. Two different prediction models yielded similar predictive power to differentiate high and low quality embryos. In terms of eventual clinical decision making, the decision tree model resulted in easy-to-interpret rules that are highly applicable in clinical practice.

  11. PERP regulates enamel formation via effects on cell-cell adhesion and gene expression.

    Science.gov (United States)

    Jheon, Andrew H; Mostowfi, Pasha; Snead, Malcolm L; Ihrie, Rebecca A; Sone, Eli; Pramparo, Tiziano; Attardi, Laura D; Klein, Ophir D

    2011-03-01

    Little is known about the role of cell-cell adhesion in the development of mineralized tissues. Here we report that PERP, a tetraspan membrane protein essential for epithelial integrity, regulates enamel formation. PERP is necessary for proper cell attachment and gene expression during tooth development, and its expression is controlled by P63, a master regulator of stratified epithelial development. During enamel formation, PERP is localized to the interface between the enamel-producing ameloblasts and the stratum intermedium (SI), a layer of cells subjacent to the ameloblasts. Perp-null mice display dramatic enamel defects, which are caused, in part, by the detachment of ameloblasts from the SI. Microarray analysis comparing gene expression in teeth of wild-type and Perp-null mice identified several differentially expressed genes during enamel formation. Analysis of these genes in ameloblast-derived LS8 cells upon knockdown of PERP confirmed the role for PERP in the regulation of gene expression. Together, our data show that PERP is necessary for the integrity of the ameloblast-SI interface and that a lack of Perp causes downregulation of genes that are required for proper enamel formation.

  12. Cell Sorting and Noise-Induced Cell Plasticity Coordinate to Sharpen Boundaries between Gene Expression Domains

    Science.gov (United States)

    2017-01-01

    A fundamental question in biology is how sharp boundaries of gene expression form precisely in spite of biological variation/noise. Numerous mechanisms position gene expression domains across fields of cells (e.g. morphogens), but how these domains are refined remains unclear. In some cases, domain boundaries sharpen through differential adhesion-mediated cell sorting. However, boundaries can also sharpen through cellular plasticity, with cell fate changes driven by up- or down-regulation of gene expression. In this context, we have argued that noise in gene expression can help cells transition to the correct fate. Here we investigate the efficacy of cell sorting, gene expression plasticity, and their combination in boundary sharpening using multi-scale, stochastic models. We focus on the formation of hindbrain segments (rhombomeres) in the developing zebrafish as an example, but the mechanisms investigated apply broadly to many tissues. Our results indicate that neither sorting nor plasticity is sufficient on its own to sharpen transition regions between different rhombomeres. Rather the two have complementary strengths and weaknesses, which synergize when combined to sharpen gene expression boundaries. PMID:28135279

  13. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu

    2014-01-01

    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

  14. Generation of hypoxanthine phosphoribosyltransferase gene knockout rabbits by homologous recombination and gene trapping through somatic cell nuclear transfer.

    Science.gov (United States)

    Yin, Mingru; Jiang, Weihua; Fang, Zhenfu; Kong, Pengcheng; Xing, Fengying; Li, Yao; Chen, Xuejin; Li, Shangang

    2015-11-02

    The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT(+/-) cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT(+/-) rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.

  15. Enrichment of human hematopoietic stem/progenitor cells facilitates transduction for stem cell gene therapy.

    Science.gov (United States)

    Baldwin, Kismet; Urbinati, Fabrizia; Romero, Zulema; Campo-Fernandez, Beatriz; Kaufman, Michael L; Cooper, Aaron R; Masiuk, Katelyn; Hollis, Roger P; Kohn, Donald B

    2015-05-01

    Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising ∼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-β(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy.

  16. Effect of TNF gene-transfected LAK cells on the ascitic liver carcinoma-bearing mice

    Institute of Scientific and Technical Information of China (English)

    Guo Liang Lou; Xue Tao Cao; Bi He Min; Wei Ping Zhang; Pei Lin Meng

    2000-01-01

    AIM To investigate the therapeutic effect of TNF gene transfected LAK cells on ascitic liver carcinoma-bearing mice.METHODS TNF gene was transfected into murine LAK cells by retrovirus. Low dose TNF gene-transfectcdLAK cells and IL-2 were i.p. injected into murine model. Cytotoxicity of gene transfected LAK cells wasstudied in vitro growth and the survival time of murine model was observed.RESULTS TNF gene-transfected LAK cells secreted higher level of TNF than that of normal LAK cells orcontrol gene-transfected LAK ceils. The in vitro growth ability and cytotoxicity of TNF gene-transfectedLAK cells were markedly inhibited by anti-TNF monoclonal antibodies. Significant therapeutic effect onascitic liver carcinoma-bearing mice was achieved.CONCLUSION TNF gene-transfected LAK cells have therapeutic effect on ascitic liver carcinoma-bearingmice.

  17. Gene editing for cell engineering: trends and applications.

    Science.gov (United States)

    Gupta, Sanjeev K; Shukla, Pratyoosh

    2016-08-18

    Gene editing with all its own advantages in molecular biology applications has made easy manipulation of various production hosts with the discovery and implementation of modern gene editing tools such as Crispr (Clustered regularly interspaced short palindromic repeats), TALENs (Transcription activator-like effector nucleases) and ZFNs (Zinc finger nucleases). With the advent of these modern tools, it is now possible to manipulate the genome of industrial production hosts such as yeast and mammalian cells which allows developing a potential and cost effective recombinant therapeutic protein. These tools also allow single editing to multiple genes for knocking-in or knocking-out of a host genome quickly in an efficient manner. A recent study on "multiplexed" gene editing revolutionized the knock-out and knock-in events of yeast and CHO, mammalian cells genome for metabolic engineering as well as high, stable, and consistent expression of a transgene encoding complex therapeutic protein such as monoclonal antibody. The gene of interest can either be integrated or deleted at single or multiple loci depending on the strategy and production requirement. This review will give a gist of all the modern tools with a brief description and advances in genetic manipulation using three major tools being implemented for the modification of such hosts with the emphasis on the use of Crispr-Cas9 for the "multiplexing gene-editing approach" for genetic manipulation of yeast and CHO mammalian hosts that ultimately leads to a fast track product development with consistent, improved product yield, quality, and thus affordability for a population at large.

  18. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  19. Mesenchymal stem cell-based gene therapy for erectile dysfunction.

    Science.gov (United States)

    Kim, J H; Lee, H J; Song, Y S

    2016-05-01

    Despite the overwhelming success of PDE5 inhibitor (PDE5I), the demand for novel pharmacotherapeutic and surgical options for ED continues to rise owing to the increased proportion of elderly individuals in the population, in addition to the growing percentage of ED patients who do not respond to PDE5I. Surgical treatment of ED is associated with many complications, thus warranting the need for nonsurgical therapies. Moreover, none of the above-mentioned treatments essentially corrects, cures or prevents ED. Although gene therapy is a promising option, many challenges and obstacles such as local inflammatory response and random transgene expression, in addition to other safety issues, limit its use at the clinical level. The use of stem cell therapy alone also has many shortcomings. To overcome these inadequacies, many scientists and clinicians are investigating new gene and stem cell therapies.

  20. Ultrastructural localization of active genes in Allium cepa cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    By using the anti-RNA/DNA hybrid antibody as the probe, we labeled and analyzed the precise transcriptional sites of active genes in Allium cepa cells in situ. The results showed that the location of labeled signals appeared in mitochondrion was the same as that in chloroplast, generally concentrated at the central matrix space where there were no cristae and thylakoids. In the extranucleolar regions of nucleus, the labeled signals of transcriptional sites were situated at the perichromatin fibrils, which decondensed and stretched out from the chromosome territories. Our results also displayed the concentrations of labeled signals in a cer-tain region of nucleus, and this means that the gene tran-scription rich region actually existed in Allium cepa cells. In nucleolus, the synthetic sites of rRNA were localized not only to the periphery of fibrillar centers but also to the DFC near FC.

  1. EVALUATION OF CYTOKINE GENE POLYMORPHISM IN B CELL LYMPHOID MALIGNANCIES

    Directory of Open Access Journals (Sweden)

    E. L. Nazarova

    2014-01-01

    Full Text Available Previous studies with some solid tumors has shown that polymorphisms of certain cytokine genes may be used as predictors of clinical outcome in the patients. It seemed important to evaluate potential correlations between production of certain pro- and anti-inflammatory cytokines and co-receptor molecules, and promoter polymorphism of the cytokine genes involved into regulation of cell proliferation, differentiation, apoptosis, lipid metabolism and blood clotting in the patients with hematological malignancies. The article contains our results concerning associations between of IL-1β, -2, -4, -10, -17, TNFα, and allelic polymorphisms of their genes in 62 patients with B cell lymphoid malignancies in an ethnically homogenous group (self-identified as Russians. We have shown that the GА and AA genotypes of the G-308A polymorphism in TNFα gene are significantly associated with increased production of this cytokine, being more common in aggressive non-Hodgkin lymphomas, more rare in multiple myeloma and in indolent non-Hodgkin lymphomas.

  2. Method for the single step introduction of a plurality of genes in microbial cells

    NARCIS (Netherlands)

    Straat, van der L.; Graaff, de L.H.

    2015-01-01

    The invention provides a method for producing a vector for introducing a plurality of genes into a host cell, the method comprising the steps of: a) providing for each gene of the plurality of genes, a DNA fragment comprising an expression cassette for expression of the gene in the host cell, wherei

  3. [VEGF gene expression in transfected human multipotent stromal cells].

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  4. Methylation of Gene CHFR Promoter in Acute Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    GONG Hui; LIU Wengli; ZHOU Jianfeng; XU Huizhen

    2005-01-01

    Summary: In order to explore whether gene CHFR was inactivated by methylation in leukemia cells, the expression of CHFR was examined before and after treatment with demethylation agent in Molt-4, Jurkat and U937 leukemia cell lines by means of RT-PCR. The methylation of promoter in Molt-4, Jurkat and U937 cells as well as 41 acute leukemia patients was analyzed by MS-PCR. The results showed that methylation of CHFR promoter was inactivated and could be reversed by treatment with a demethylating agent in Molt-4, Jurkat and U937. CHFR promoter methylation was detected in 39 % of acute leukemia patients. There was no difference in incidence of CHFR promoter methylation between acute myelocytic leukemia and acute lymphocytic leukemia. In conclusion, CHFR is frequently inactivated in acute leukemia and is a good candidate for the leukemia supper gene. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in acute leukemia. Moreover, the methylation of gene CHFR appears to be a good index with which to predict the sensitivity of acute leukemia to microtubule inhibitors.

  5. MGFM: a novel tool for detection of tissue and cell specific marker genes from microarray gene expression data.

    Science.gov (United States)

    El Amrani, Khadija; Stachelscheid, Harald; Lekschas, Fritz; Kurtz, Andreas; Andrade-Navarro, Miguel A

    2015-08-28

    Identification of marker genes associated with a specific tissue/cell type is a fundamental challenge in genetic and cell research. Marker genes are of great importance for determining cell identity, and for understanding tissue specific gene function and the molecular mechanisms underlying complex diseases. We have developed a new bioinformatics tool called MGFM (Marker Gene Finder in Microarray data) to predict marker genes from microarray gene expression data. Marker genes are identified through the grouping of samples of the same type with similar marker gene expression levels. We verified our approach using two microarray data sets from the NCBI's Gene Expression Omnibus public repository encompassing samples for similar sets of five human tissues (brain, heart, kidney, liver, and lung). Comparison with another tool for tissue-specific gene identification and validation with literature-derived established tissue markers established functionality, accuracy and simplicity of our tool. Furthermore, top ranked marker genes were experimentally validated by reverse transcriptase-polymerase chain reaction (RT-PCR). The sets of predicted marker genes associated with the five selected tissues comprised well-known genes of particular importance in these tissues. The tool is freely available from the Bioconductor web site, and it is also provided as an online application integrated into the CellFinder platform ( http://cellfinder.org/analysis/marker ). MGFM is a useful tool to predict tissue/cell type marker genes using microarray gene expression data. The implementation of the tool as an R-package as well as an application within CellFinder facilitates its use.

  6. Transcriptional regulation of cathelicidin genes in chicken bone marrow cells.

    Science.gov (United States)

    Lee, Sang In; Jang, Hyun June; Jeon, Mi-hyang; Lee, Mi Ock; Kim, Jeom Sun; Jeon, Ik-Soo; Byun, Sung June

    2016-04-01

    Cathelicidins form a family of vertebrate-specific immune molecules with an evolutionarily conserved gene structure. We analyzed the expression patterns of cathelicidin genes (CAMP, CATH3, and CATHB1) in chicken bone marrow cells (BMCs) and chicken embryonic fibroblasts (CEFs). We found that CAMP and CATHB1 were significantly up-regulated in BMCs, whereas the expression of CATH3 did not differ significantly between BMCs and CEFs. To study the mechanism underlying the up-regulation of cathelicidin genes in BMCs, we predicted the transcription factors (TFs) that bind to the 5'-flanking regions of cathelicidin genes. CEBPA, EBF1, HES1, MSX1, and ZIC3 were up-regulated in BMCs compared to CEFs. Subsequently, when a siRNA-mediated knockdown assay was performed for MSX1, the expression of CAMP and CATHB1 was decreased in BMCs. We also showed that the transcriptional activity of the CAMP promoter was decreased by mutation of the MSX1-binding sites present within the 5'-flanking region of CAMP. These results increase our understanding of the regulatory mechanisms controlling cathelicidin genes in BMCs.

  7. MGMT enrichment and second gene co-expression in hematopoietic progenitor cells using separate or dual-gene lentiviral vectors.

    Science.gov (United States)

    Roth, Justin C; Alberti, Michael O; Ismail, Mourad; Lingas, Karen T; Reese, Jane S; Gerson, Stanton L

    2015-01-22

    The DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT) allows efficient in vivo enrichment of transduced hematopoietic stem cells (HSC). Thus, linking this selection strategy to therapeutic gene expression offers the potential to reconstitute diseased hematopoietic tissue with gene-corrected cells. However, different dual-gene expression vector strategies are limited by poor expression of one or both transgenes. To evaluate different co-expression strategies in the context of MGMT-mediated HSC enrichment, we compared selection and expression efficacies in cells cotransduced with separate single-gene MGMT and GFP lentivectors to those obtained with dual-gene vectors employing either encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) or foot and mouth disease virus (FMDV) 2A elements for co-expression strategies. Each strategy was evaluated in vitro and in vivo using equivalent multiplicities of infection (MOI) to transduce 5-fluorouracil (5-FU) or Lin(-)Sca-1(+)c-kit(+) (LSK)-enriched murine bone marrow cells (BMCs). The highest dual-gene expression (MGMT(+)GFP(+)) percentages were obtained with the FMDV-2A dual-gene vector, but half of the resulting gene products existed as fusion proteins. Following selection, dual-gene expression percentages in single-gene vector cotransduced and dual-gene vector transduced populations were similar. Equivalent MGMT expression levels were obtained with each strategy, but GFP expression levels derived from the IRES dual-gene vector were significantly lower. In mice, vector-insertion averages were similar among cells enriched after dual-gene vectors and those cotransduced with single-gene vectors. These data demonstrate the limitations and advantages of each strategy in the context of MGMT-mediated selection, and may provide insights into vector design with respect to a particular therapeutic gene or hematologic defect.

  8. Overview of gene therapy clinical progress including cancer treatment with gene-modified T cells.

    Science.gov (United States)

    Brenner, Malcolm K; Okur, Fatma V

    2009-01-01

    It is now twenty years since the first legal gene transfer studies were approved, and there has been considerable disappointment in the slow rate of progress that followed the initial studies. Gradually, however, as the limitations of available vectors are acknowledged and overcome, and with advances in our understanding of the molecular and cell biology of genetic diseases and of cancer, unequivocal successes are now being reported. In this paper we describe the remaining major roadblocks to successful gene therapy and outline approaches to overcome them. We also illustrate how genetically modified immune system cells are already being used for the effective treatment of hematological and other malignancies, and how these approaches are being modified so that they can be effective in treating a broader range of malignancies.

  9. In search of a suitable reference gene for normalization of gene expression in MDV-infected chicken cells

    Science.gov (United States)

    Marek’s disease (MD) is a contagious lymphoproliferative disease of domestic chickens caused by a highly cell-associated alpha-herpesvirus, MD virus (MDV). The choice of an appropriate housekeeping gene as an endogenous reference gene is an essential requirement for relative quantification of gene ...

  10. Differential gene expression in stromal cells of human giant cell tumor of bone.

    Science.gov (United States)

    Wuelling, M; Delling, G; Kaiser, E

    2004-12-01

    Giant cell tumor (GCT) offers a unique model for the hematopoietic-stromal cell interaction in human bone marrow. Evidence has been presented that GCT stromal cells (GCTSCs) promote accumulation, size and activity of the giant cells. Although GCTSCs are considered the neoplastic component of GCT, little is known about their genetic basis and, to date, a tumor-specific gene expression pattern has not been characterized. Mesenchymal stem cells (MSCs) have been identified as the origin of the GCT neoplastic stromal cell. Using state of the art array technology, expression profiling was applied to enriched stromal cell populations from five different GCTs and two primary MSCs as controls. Of the 29 differentially expressed genes found, 25 showed an increased expression. Differential mRNA expression was verified by real-time polymerase chain reaction analysis of 10 selected genes, supporting the validity of cDNA arrays as a tool to identify tumor-related genes in GCTSCs. Increased expression of two oncogenes, JUN and NME2, was substantiated at the protein level, utilizing immunohistochemical evaluation of GCT sections and Western-blot analysis. Increased phosphorylation of JUN Ser-63 was also found.

  11. Expression of insulin-like growth factor family genes in clear cell renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Ryszard Braczkowski

    2016-06-01

    Full Text Available Aim of the study : Despite significant progress in the pathology of clear cell renal cell carcinoma (ccRCC, diagnostic and predictive factors of major importance have not been discovered. Some hopes are associated with insulin-like growth factors. The aim of the study was to compare the expression of genes for insulin-like growth factor family in tumours and in tissue of kidneys without cancer. Material and methods : Fifty-two patients years with clear cell renal cell cancer were qualified to the study group; patients nephrectomised because of hydronephrosis were included in the control group. Expression of genes were evaluated by RT-PCR. Results : Expression of IGFR-1 gene in tumour accounts for about 60% of cases. The incidence is higher than in corresponding adjacent non-cancerous kidney tissues and higher (but with no statistical significance than in kidney without cancer. Expression of IGFR-2 gene in tumours has not been established. The incidence of the expression in corresponding adjacent non-cancerous kidney tissues is small. Expression of this gene has been present in all specimens from kidneys without cancer. Expression of IGFBP-3 gene ascertained in all (except four cases of ccRCC and in the majority of clippings from adjacent tissue. It was not found in kidneys from the control group. IGF-1, IGF-2, and IGFR-1 mRNA copy numbers in ccRCC were higher than in the material from the control group

  12. Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

    Science.gov (United States)

    Ruszymah, B H I; Izham, B A Azrul; Heikal, M Y Mohd; Khor, S F; Fauzi, M B; Aminuddin, B S

    2011-12-01

    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.

  13. From the Cover: Design of artificial cell-cell communication using gene and metabolic networks

    Science.gov (United States)

    Bulter, Thomas; Lee, Sun-Gu; Waichun Wong, Wilson; Fung, Eileen; Connor, Michael R.; Liao, James C.

    2004-02-01

    Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell-cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell-cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling pH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp2 promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model.

  14. In Vitro Antioxidant-Activity Evaluation of Gallic-Acid-Grafted Chitosan Conjugate Synthesized by Free-Radical-Induced Grafting Method.

    Science.gov (United States)

    Hu, Qiaobin; Wang, Taoran; Zhou, Mingyong; Xue, Jingyi; Luo, Yangchao

    2016-07-27

    The major objective of this work was to develop a green and facile process to prepare gallic acid-chitosan conjugate and comprehensively evaluate the physicochemical properties and biological activities of an as-prepared water-soluble chitosan derivative. A free-radical-induced grafting approach using an ascorbic acid-hydrogen peroxide redox pair was adopted. The obtained conjugate was characterized by Fourier transform infrared spectroscopy, UV-vis, X-ray diffraction, and pKa analysis. The antioxidant activities were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6)-sulphonic acid (ABTS), reducing power, and oxygen-radical antioxidant-capacity assays. The results showed that the mass ratio of gallic acid to chitosan played a vital role in determining the grafting degree and ζ potential of the conjugates, with the ratio of 0.5:1 being the optimal ratio that resulted in the highest grafting degree. The antioxidant assays demonstrated that conjugation significantly improved the antioxidant activities, being dramatically higher than that of free chitosan. It was notable that the DPPH- and ABTS-scavenging activities of conjugate at 0.4 mg/mL reached the same level as the free gallic acid at the equivalent concentration. Our study demonstrated a green and facile synthesis approach to preparing a novel water-soluble chitosan derivative that may have promising potentials in the food industry.

  15. Measuring T cell receptor and T cell gene expression diversity in antigen-responsive human CD4+ T cells.

    Science.gov (United States)

    Eugster, Anne; Lindner, Annett; Heninger, Anne-Kristin; Wilhelm, Carmen; Dietz, Sevina; Catani, Mara; Ziegler, Anette-G; Bonifacio, Ezio

    2013-12-31

    T cells have diversity in TCR, epitope recognition, and cytokine production, and can be used for immune monitoring. Furthermore, clonal expansion of TCR families in disease may provide opportunities for TCR-directed therapies. We developed methodology for sequencing expressed genes of TCR alpha and beta chains from single cells and applied this to vaccine (tetanus-toxoid)-responsive CD4(+) T cells. TCR alpha and beta chains were both successfully sequenced in 1309 (43%) of 3038 CD4(+) T cells yielding 677 different receptors. TRAV and TRBV gene usage differed between tetanus-toxoid-responsive and non-responsive cells (p=0.004 and 0.0002), and there was extensive TCR diversity in tetanus-toxoid-responsive cells within individuals. Identical TCRs could be recovered in different samples from the same subject: TCRs identified after booster vaccination were frequent in pre-booster memory T cells (31% of pre-booster TCR), and also identified in pre-booster vaccination naïve cells (6.5%). No TCR was shared between subjects, but tetanus toxoid-responsive cells sharing one of their TCR chains were observed within and between subjects. Coupling single-cell gene expression profiling to TCR sequencing revealed examples of distinct cytokine profiles in cells bearing identical TCR. Novel molecular methodology demonstrates extensive diversity of Ag-responsive CD4(+) T cells within and between individuals. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. [Experimental gene therapy using p21/WAF1 gene in esophageal squamous cell carcinoma--adenovirus infection and gene gun technology].

    Science.gov (United States)

    Fujii, T; Tanaka, Y; Tanaka, T; Matono, S; Sueyoshi, S; Fujita, H; Shirouzu, K; Kato, S; Yamana, H

    2001-10-01

    p21/WAF1 (p21) inhibits the activity of the cyclin/cdk complex and controls the G1 to S cell phase transition. In the present study, we used a recombinant adenoviral approach and gene gun technology to introduce p21 into esophageal cancer cells in order to assess the effect of p21 on cell growth. Infection with the p21 adenovirus (AdV) using gene gun technology resulted in inhibition of TE9 and KE3 cell growth. The levels of involucrin, which is a marker of squamous epithelium differentiation, markedly increased at 48 h and 72 h after p21 AdV infection in TE9 cells. These results indicate that p21 plays an important role in esophageal cancer cell proliferation. Overexpression of the p21 gene can inhibit cell growth and induce differentiation in esophageal cancer cells. p21 gene therapy may prove beneficial in the treatment of esophageal cancer.

  17. Treating hearing disorders with cell and gene therapy

    Science.gov (United States)

    Gillespie, Lisa N.; Richardson, Rachael T.; Nayagam, Bryony A.; Wise, Andrew K.

    2014-12-01

    Hearing loss is an increasing problem for a substantial number of people and, with an aging population, the incidence and severity of hearing loss will become more significant over time. There are very few therapies currently available to treat hearing loss, and so the development of new therapeutic strategies for hearing impaired individuals is of paramount importance to address this unmet clinical need. Most forms of hearing loss are progressive in nature and therefore an opportunity exists to develop novel therapeutic approaches to slow or halt hearing loss progression, or even repair or replace lost hearing function. Numerous emerging technologies have potential as therapeutic options. This paper details the potential of cell- and gene-based therapies to provide therapeutic agents to protect sensory and neural cells from various insults known to cause hearing loss; explores the potential of replacing lost sensory and nerve cells using gene and stem cell therapy; and describes the considerations for clinical translation and the challenges that need to be overcome.

  18. Cell of origin associated classification of B-cell malignancies by gene signatures of the normal B-cell hierarchy.

    Science.gov (United States)

    Johnsen, Hans Erik; Bergkvist, Kim Steve; Schmitz, Alexander; Kjeldsen, Malene Krag; Hansen, Steen Møller; Gaihede, Michael; Nørgaard, Martin Agge; Bæch, John; Grønholdt, Marie-Louise; Jensen, Frank Svendsen; Johansen, Preben; Bødker, Julie Støve; Bøgsted, Martin; Dybkær, Karen

    2014-06-01

    Recent findings have suggested biological classification of B-cell malignancies as exemplified by the "activated B-cell-like" (ABC), the "germinal-center B-cell-like" (GCB) and primary mediastinal B-cell lymphoma (PMBL) subtypes of diffuse large B-cell lymphoma and "recurrent translocation and cyclin D" (TC) classification of multiple myeloma. Biological classification of B-cell derived cancers may be refined by a direct and systematic strategy where identification and characterization of normal B-cell differentiation subsets are used to define the cancer cell of origin phenotype. Here we propose a strategy combining multiparametric flow cytometry, global gene expression profiling and biostatistical modeling to generate B-cell subset specific gene signatures from sorted normal human immature, naive, germinal centrocytes and centroblasts, post-germinal memory B-cells, plasmablasts and plasma cells from available lymphoid tissues including lymph nodes, tonsils, thymus, peripheral blood and bone marrow. This strategy will provide an accurate image of the stage of differentiation, which prospectively can be used to classify any B-cell malignancy and eventually purify tumor cells. This report briefly describes the current models of the normal B-cell subset differentiation in multiple tissues and the pathogenesis of malignancies originating from the normal germinal B-cell hierarchy.

  19. Heat shock genes – integrating cell survival and death

    Indian Academy of Sciences (India)

    Richa Arya; Moushami Mallik; Subhash C Lakhotia

    2007-04-01

    Heat shock induced gene expression and other cellular responses help limit the damage caused by stress and thus facilitate cellular recovery. Cellular damage also triggers apoptotic cell death through several pathways. This paper briefly reviews interactions of the major heat shock proteins with components of the apoptotic pathways. Hsp90, which acts as a chaperone for unstable signal transducers to keep them poised for activation, interacts with RIP and Akt and promotes NF-B mediated inhibition of apoptosis; in addition it also blocks some steps in the apoptotic pathways. Hsp70 is mostly anti-apoptotic and acts at several levels like inhibition of translocation of Bax into mitochondria, release of cytochrome c from mitochondria, formation of apoptosome and inhibition of activation of initiator caspases. Hsp70 also modulates JNK, NF-B and Akt signaling pathways in the apoptotic cascade. In contrast, Hsp60 has both anti- and pro-apoptotic roles. Cytosolic Hsp60 prevents translocation of the pro-apoptotic protein Bax into mitochondria and thus promotes cell survival but it also promotes maturation of procaspase-3, essential for caspase mediated cell death. Our recent in vivo studies show that RNAi for the Hsp60D in Drosophila melanogaster prevents induced apoptosis. Hsp27 exerts its anti-apoptotic influence by inhibiting cytochrome c and TNF-mediated cell death. crystallin suppresses caspase-8 and cytochrome c mediated activation of caspase-3. Studies in our laboratory also reveal that absence or reduced levels of the developmentally active as well as stress induced non-coding hsr transcripts, which are known to sequester diverse hnRNPs and related nuclear RNA-binding proteins, block induced apoptosis in Drosophila. Modulation of the apoptotic pathways by Hsps reflects their roles as ``weak links” between various ``hubs” in cellular networks. On the other hand, non-coding RNAs, by virtue of their potential to bind with multiple proteins, can act as ``hubs” in

  20. BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways.

    Science.gov (United States)

    Passarinho, Paul; Ketelaar, Tijs; Xing, Meiqing; van Arkel, Jeroen; Maliepaard, Chris; Hendriks, Mieke Weemen; Joosen, Ronny; Lammers, Michiel; Herdies, Lydia; den Boer, Bart; van der Geest, Lonneke; Boutilier, Kim

    2008-10-01

    Ectopic expression of the Brassica napus BABY BOOM (BBM) AP2/ERF transcription factor is sufficient to induce spontaneous cell proliferation leading primarily to somatic embryogenesis, but also to organogenesis and callus formation. We used DNA microarray analysis in combination with a post-translationally regulated BBM:GR protein and cycloheximide to identify target genes that are directly activated by BBM expression in Arabidopsis seedlings. We show that BBM activated the expression of a largely uncharacterized set of genes encoding proteins with potential roles in transcription, cellular signaling, cell wall biosynthesis and targeted protein turnover. A number of the target genes have been shown to be expressed in meristems or to be involved in cell wall modifications associated with dividing/growing cells. One of the BBM target genes encodes an ADF/cofilin protein, ACTIN DEPOLYMERIZING FACTOR9 (ADF9). The consequences of BBM:GR activation on the actin cytoskeleton were followed using the GFP:FIMBRIN ACTIN BINDING DOMAIN2 (GFP:FABD) actin marker. Dexamethasone-mediated BBM:GR activation induced dramatic changes in actin organization resulting in the formation of dense actin networks with high turnover rates, a phenotype that is consistent with cells that are rapidly undergoing cytoplasmic reorganization. Together the data suggest that the BBM transcription factor activates a complex network of developmental pathways associated with cell proliferation and growth.

  1. ET-67SUICIDE GENE THERAPY FOR GLIOMA USING MULTILINEAGE-DEFFERENTIATING STRESS ENDURING (MUSE) CELLS

    OpenAIRE

    Yamasaki, Tomohiro; Wakao, Shohei; Kawaji, Hiroshi; Suzuki, Tomo; Kamio, Yoshinobu; AMANO, SHINJI; Sameshima, Tetsuro; Sakai, Naoto; TOKUYAMA, TSUTOMU; Dezawa, Mari; Namba, Hiroki

    2014-01-01

    INTRODUCTION: We have been investigating cell-based glioma gene therapy using various kinds of stem cells transduced with the herpes simplex virus thymidine kinase gene (HSVtk). In our previous study, we used SSEA3/CD105 double-positive multilineage-differentiating stress-enduring (Muse) cells transduced with HSVtk (Muse-tk cells) as the vehicle for HSVtk/ganciclovir (GCV) gene therapy. We demonstrated a potent in vitro tumoricidal bystander effect for various glioma cells. In the present stu...

  2. Persistent donor cell gene expression among human induced pluripotent stem cells contributes to differences with human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhumur Ghosh

    Full Text Available Human induced pluripotent stem cells (hiPSCs generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs, as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.

  3. Stem Leydig Cell Differentiation: Gene Expression During Development of the Adult Rat Population of Leydig Cells1

    Science.gov (United States)

    Stanley, Erin L.; Johnston, Daniel S.; Fan, Jinjiang; Papadopoulos, Vassilios; Chen, Haolin; Ge, Ren-Shan; Zirkin, Barry R.; Jelinsky, Scott A.

    2011-01-01

    ABSTRACT Leydig cells are the testosterone-producing cells in the adult male. Adult Leydig cells (ALCs) develop from stem Leydig cells (SLCs) through at least two intermediate cells, progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). Microarray gene expression was used to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to that in a well-established stem cell, bone marrow stem cells (BSCs). We show that the pattern of gene expression by SLCs was more similar to the expression by BSCs, an established stem cell outside the male reproductive tract, than to any of the cells in the Leydig cell developmental lineage. These results indicated that the SLCs have many of the molecular characteristics of other stem cells. Pathway analysis indicated that development of Leydig cells from SLCs to PLCs was associated with decreased expression of genes related to adhesion and increased expression of genes related to steroidogenesis. Gene expression changes between PLCs and ILCs and between ILCs and ALCs were relatively minimal, suggesting that these cells are highly similar. In contrast, gene expression changes between SLCs and ALCs were quite distinct. PMID:21832170

  4. Cell culture density affects the stemness gene expression of adipose tissue-derived mesenchymal stem cells.

    Science.gov (United States)

    Kim, Dae Seong; Lee, Myoung Woo; Lee, Tae-Hee; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

    2017-03-01

    The results of clinical trials using mesenchymal stem cells (MSCs) are controversial due to the heterogeneity of human MSCs and differences in culture conditions. In this regard, it is important to identify gene expression patterns according to culture conditions, and to determine how the cells are expanded and when they should be clinically used. In the current study, stemness gene expression was investigated in adipose tissue-derived MSCs (AT-MSCs) harvested following culture at different densities. AT-MSCs were plated at a density of 200 or 5,000 cells/cm(2). After 7 days of culture, stemness gene expression was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The proliferation rate of AT-MSCs harvested at a low density (~50% confluent) was higher than that of AT-MSCs harvested at a high density (~90% confluent). Although there were differences in the expression levels of stemness gene, such as octamer-binding transcription factor 4, nanog homeobox (Nanog), SRY-box 2, Kruppel like factor 4, v-myc avian myelocytomatosis viral oncogene homolog (c-Myc), and lin-28 homolog A, in the AT-MSCs obtained from different donors, RT-qPCR analysis demonstrated differential gene expression patterns according to the cell culture density. Expression levels of stemness genes, particularly Nanog and c-Myc, were upregulated in AT-MSCs harvested at a low density (~50% confluent) in comparison to AT-MSCs from the same donor harvested at a high density (~90% confluent). These results imply that culture conditions, such as the cell density at harvesting, modulate the stemness gene expression and proliferation of MSCs.

  5. Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development.

    Science.gov (United States)

    Chen, Liang-Yu; Willis, William D; Eddy, Edward M

    2016-02-16

    Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo.

  6. Free radicals induced by sunlight in different spectral regions - in vivo versus ex vivo study.

    Science.gov (United States)

    Lohan, Silke B; Müller, Robert; Albrecht, Stephanie; Mink, Kathrin; Tscherch, Kathrin; Ismaeel, Fakher; Lademann, Jürgen; Rohn, Sascha; Meinke, Martina C

    2016-05-01

    Sunlight represents an exogenous factor stimulating formation of free radicals which can induce cell damage. To assess the effect of the different spectral solar regions on the development of free radicals in skin, in vivo electron paramagnetic resonance (EPR) investigations with human volunteers and ex vivo studies on excised human and porcine skin were carried out. For all skin probes, the ultraviolet (UV) spectral region stimulates the most intensive radical formation, followed by the visible (VIS) and the near infrared (NIR) regions. A comparison between the different skin models shows that for UV light, the fastest and highest production of free radicals could be detected in vivo, followed by excised porcine and human skin. The same distribution pattern was found for the VIS/NIR spectral regions, whereby the differences in radical formation between in vivo and ex vivo were less pronounced. An analysis of lipid composition in vivo before and after exposure to UV light clearly showed modifications in several skin lipid components; a decrease of ceramide subclass [AP2] and an increase of ceramide subclass [NP2], sodium cholesterol sulphate and squalene (SQ) were detectable. In contrast, VIS/NIR irradiation led to an increase of ceramides [AP2] and SCS, and a decrease of SQ. These results, which are largely comparable for the different skin models investigated in vivo and ex vivo, indicate that radiation exposure in different spectral regions strongly influences radical production in skin and also results in changes in skin lipid composition, which is essential for barrier function.

  7. Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells.

    Science.gov (United States)

    Tansriratanawong, Kallapat; Tamaki, Yuichi; Ishikawa, Hiroshi; Sato, Soh

    2014-10-01

    In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPARγ2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration.

  8. Induction in myeloid leukemic cells of genes that are expressed in different normal tissues

    OpenAIRE

    2005-01-01

    Using DNA microarray and cluster analysis of expressed genes in a cloned line (M1-t-p53) of myeloid leukemic cells, we have analyzed the expression of genes that are preferentially expressed in different normal tissues. Clustering of 547 highly expressed genes in these leukemic cells showed 38 genes preferentially expressed in normal hematopoietic tissues and 122 other genes preferentially expressed in different normal non-hematopoietic tissues including neuronal tissues, muscle, liver and te...

  9. Adenovirus-mediated gene transfer to tumor cells.

    Science.gov (United States)

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting.

  10. Gene expression in epithelial cells in response to pneumovirus infection

    Directory of Open Access Journals (Sweden)

    Rosenberg Helene F

    2001-05-01

    Full Text Available Abstract Respiratory syncytial virus (RSV and pneumonia virus of mice (PVM are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner.

  11. Gene amplification during differentiation of mammalian neural stem cells in vitro and in vivo.

    Science.gov (United States)

    Fischer, Ulrike; Backes, Christina; Raslan, Abdulrahman; Keller, Andreas; Meier, Carola; Meese, Eckart

    2015-03-30

    In development of amphibians and flies, gene amplification is one of mechanisms to increase gene expression. In mammalian cells, gene amplification seems to be restricted to tumorigenesis and acquiring of drug-resistance in cancer cells. Here, we report a complex gene amplification pattern in mouse neural progenitor cells during differentiation with approximately 10% of the genome involved. Half of the amplified mouse chromosome regions overlap with amplified regions previously reported in human neural progenitor cells, indicating conserved mechanisms during differentiation. Using fluorescence in situ hybridization, we verified the amplification in single cells of primary mouse mesencephalon E14 (embryonic stage) neurosphere cells during differentiation. In vivo we confirmed gene amplifications of the TRP53 gene in cryosections from mouse embryos at stage E11.5. Gene amplification is not only a cancer-related mechanism but is also conserved in evolution, occurring during differentiation of mammalian neural stem cells.

  12. A systematic study on drug-response associated genes using baseline gene expressions of the Cancer Cell Line Encyclopedia

    Science.gov (United States)

    Liu, Xiaoming; Yang, Jiasheng; Zhang, Yi; Fang, Yun; Wang, Fayou; Wang, Jun; Zheng, Xiaoqi; Yang, Jialiang

    2016-03-01

    We have studied drug-response associated (DRA) gene expressions by applying a systems biology framework to the Cancer Cell Line Encyclopedia data. More than 4,000 genes are inferred to be DRA for at least one drug, while the number of DRA genes for each drug varies dramatically from almost 0 to 1,226. Functional enrichment analysis shows that the DRA genes are significantly enriched in genes associated with cell cycle and plasma membrane. Moreover, there might be two patterns of DRA genes between genders. There are significantly shared DRA genes between male and female for most drugs, while very little DRA genes tend to be shared between the two genders for a few drugs targeting sex-specific cancers (e.g., PD-0332991 for breast cancer and ovarian cancer). Our analyses also show substantial difference for DRA genes between young and old samples, suggesting the necessity of considering the age effects for personalized medicine in cancers. Lastly, differential module and key driver analyses confirm cell cycle related modules as top differential ones for drug sensitivity. The analyses also reveal the role of TSPO, TP53, and many other immune or cell cycle related genes as important key drivers for DRA network modules. These key drivers provide new drug targets to improve the sensitivity of cancer therapy.

  13. Utilizing cell-matrix interactions to modulate gene transfer to stem cells inside hyaluronic acid hydrogels.

    Science.gov (United States)

    Gojgini, Shiva; Tokatlian, Talar; Segura, Tatiana

    2011-10-01

    The effective delivery of DNA locally would increase the applicability of gene therapy in tissue regeneration, where diseased tissue is to be repaired in situ. One promising approach is to use hydrogel scaffolds to encapsulate and deliver plasmid DNA in the form of nanoparticles to the diseased tissue, so that cells infiltrating the scaffold are transfected to induce regeneration. This study focuses on the design of a DNA nanoparticle-loaded hydrogel scaffold. In particular, this study focuses on understanding how cell-matrix interactions affect gene transfer to adult stem cells cultured inside matrix metalloproteinase (MMP) degradable hyaluronic acid (HA) hydrogel scaffolds. HA was cross-linked to form a hydrogel material using a MMP degradable peptide and Michael addition chemistry. Gene transfer inside these hydrogel materials was assessed as a function of polyplex nitrogen to phosphate ratio (N/P = 5 to 12), matrix stiffness (100-1700 Pa), RGD (Arg-Gly-Asp) concentration (10-400 μM), and RGD presentation (0.2-4.7 RGDs per HA molecule). All variables were found to affect gene transfer to mouse mensenchymal stem cells culture inside the DNA loaded hydrogels. As expected, higher N/P ratios lead to higher gene transfer efficiency but also higher toxicity; softer hydrogels resulted in higher transgene expression than stiffer hydrogels, and an intermediate RGD concentration and RGD clustering resulted in higher transgene expression. We believe that the knowledge gained through this in vitro model can be utilized to design better scaffold-mediated gene delivery for local gene therapy.

  14. Analysis of Gene Expression in the K562-n High Tumorigenitic Human Leukemia Cell Line

    Institute of Scientific and Technical Information of China (English)

    Shuqing Lü; Xiaoping Xu; Fang Xia; JianMin Wang

    2005-01-01

    OBJECTIVE The human leukemia K562-n cell line displays much higher tumorigenic actively in nude mice compared with its parental K562 cell line. The molecular mechanism of the differences in tumorigenicity between K562-n and K562 in nude mice was examined.METHODS The differences in gene expression between K562 and K562-n cells were analyzed by using cDNA microarrays.RESULTS Among the12,800 genes examined, there was a significant difference in expression of 139 genes between K562-n and K562 cells.Eighty-five of these genes have been registered in the GeneBank and 54are unknown. The genes accessible from the GeneBank include:1)oncogenes and tumor-supressor genes; 2) genes related to transcription regulation, the cell cycle and apoptosis; 3) genes related to the cytoskeleton and cytokinetics; 4) genes related to metabolism and transport; 5) genes related to immune function. There were also some differently expressed genes with mixed functions.CONCLUSION There are many genes differentially expressed between K562-n and K562 cells .The high tumorigenicity of the human leukemia K562-n cell line in nude mice might be related to its specific geneexpression profile.

  15. HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Hongzhu LU; Jianhua ZHOU

    2008-01-01

    The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

  16. Differential expression of the klf6 tumor suppressor gene upon cell damaging treatments in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Gehrau, Ricardo C.; D' Astolfo, Diego S.; Andreoli, Veronica [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Bocco, Jose L., E-mail: jbocco@fcq.unc.edu.ar [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Koritschoner, Nicolas P. [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina)

    2011-02-10

    The mammalian Krueppel-like factor 6 (KLF6) is involved in critical roles such as growth-related signal transduction, cell proliferation and differentiation, development, apoptosis and angiogenesis. Also, KLF6 appears to be an emerging key factor during cancer development and progression. Its expression is thoroughly regulated by several cell-damaging stimuli. DNA damaging agents at lethal concentrations induce a p53-independent down-regulation of the klf6 gene. To investigate the impact of external stimuli on human klf6 gene expression, its mRNA level was analyzed using a cancer cell line profiling array system, consisting in an assortment of immobilized cDNAs from multiple cell lines treated with several cell-damaging agents at growth inhibitory concentrations (IC{sub 50}). Cell-damaging agents affected the klf6 expression in 62% of the cDNA samples, though the expression pattern was not dependent on the cell origin type. Interestingly, significant differences (p < 0.0001) in KLF6 mRNA levels were observed depending on the cellular p53 status upon cell damage. KLF6 expression was significantly increased in 63% of p53-deficient cells (122/195). Conversely, KLF6 mRNA level decreased nearly 4 fold in more than 70% of p53+/+ cells. In addition, klf6 gene promoter activity was down-regulated by DNA damaging agents in cells expressing the functional p53 protein whereas it was moderately increased in the absence of functional p53. Consistent results were obtained for the endogenous KLF6 protein level. Results indicate that human klf6 gene expression is responsive to external cell damage mediated by IC{sub 50} concentrations of physical and chemical stimuli in a p53-dependent manner. Most of these agents are frequently used in cancer therapy. Induction of klf6 expression in the absence of functional p53 directly correlates with cell death triggered by these compounds, whereas it is down-regulated in p53+/+ cells. Hence, klf6 expression level could represent a valuable

  17. Retroviral transfer of the nlsLacZ gene into human CD34+ cell populations and into TF-1 cells: future prospects in gene therapy.

    Science.gov (United States)

    Bagnis, C; Gravis, G; Imbert, A M; Herrera, D; Allario, T; Galindo, R; Lopez, M; Pavon, C; Sempere, C; Mannoni, P

    1994-11-01

    Few data are available concerning behavior of reimplanted human hematopoietic cells after autologous stem cell transplantation. This paper reports the possibility to transfer gene markers coding for beta-galactosidase (beta-Gal) activity by retroviral vectors into a human leukemic growth factor-dependent cell line, TF-1, and into human hematopoietic progenitors isolated from peripheral blood or bone marrow. Using various combinations of retroviral vectors and packaging cell lines, we demonstrated high expression of a bacterial beta-Gal activity induced by the LacZ gene, the nlsLacZ gene, or the Sh-ble/LacZ gene, in human hematopoietic cells. The expression of the nlsLacZ construct was stable until the end of the culture in infected CD34+ cell-enriched cell populations, and a slow decrease of transgene expression was observed in a transduced TF-1 cell population during a 1-year long-term culture. Data obtained with the nlsLacZ gene demonstrate that both retroviral transfer and corresponding gene expression were not found to modify the pattern of cell proliferation and differentiation. These results open interesting prospectives for the use of the nlsLacZ gene to mark and follow the fate of progenitor cells isolated from patients with cancers prior to reimplantation.

  18. Identification of cell cycle-regulated genes by convolutional neural network.

    Science.gov (United States)

    Liu, Chenglin; Cui, Peng; Huang, Tao

    2017-04-17

    The cell cycle-regulated genes express periodically with the cell cycle stages, and the identification and study of these genes can provide a deep understanding of the cell cycle process. Large false positives and low overlaps are big problems in cell cycle-regulated gene detection. Here, a computational framework called DLGene was proposed for cell cycle-regulated gene detection. It is based on the convolutional neural network, a deep learning algorithm representing raw form of data pattern without assumption of their distribution. First, the expression data was transformed to categorical state data to denote the changing state of gene expression, and four different expression patterns were revealed for the reported cell cycle-regulated genes. Then, DLGene was applied to discriminate the non-cell cycle gene and the four subtypes of cell cycle genes. Its performances were compared with six traditional machine learning methods. At last, the biological functions of representative cell cycle genes for each subtype were analyzed. Our method showed better and more balanced performance of sensitivity and specificity comparing to other machine learning algorithms. The cell cycle genes had very different expression pattern with non-cell cycle genes and among the cell-cycle genes, there were four subtypes. Our method not only detects the cell cycle genes, but also describes its expression pattern, such as when its highest expression level is reached and how it changes with time. For each type, we analyzed the biological functions of the representative genes and such results provided novel insight of the cell cycle mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri

    Directory of Open Access Journals (Sweden)

    Hallmann Armin

    2006-12-01

    Full Text Available Abstract Background The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there are ongoing genome projects both in Volvox carteri and in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluate the potential of real-time RT-PCR for quantifying gene transcript levels. Results Here we analyze a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contains previously known genes with unknown localization of cellular expression, 28 novel genes which are described in this study for the first time, and a few known, cell-type specific genes as a control. The respective gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provide expression data for all these genes. Conclusion The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox, which can be extended to a much larger number of genes or to developmental or metabolic mutants. Our expression data also provide a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes.

  20. Ex-Vivo Gene Therapy Using Lentiviral Mediated Gene Transfer Into Umbilical Cord Blood Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Hanieh Jalali

    2016-02-01

    Full Text Available Background Introduction of therapeutic genes into the injured site of nervous system can be achieved using transplantation of cellular vehicles containing desired gene. To transfer exogenous genes into the cellular vehicles, lentiviral vectors are one of interested vectors because of advantages such high transduction efficiency of dividing and non-dividing cells. Unrestricted somatic stem cells are subclasses of umbilical cord blood derived stem cells which are appreciate candidates to use as cellular vehicles for ex vivo gene therapy of nervous system. Objectives In current study we investigated the effect of lentiviral vector transduction on the neuronal related features of unrestricted somatic stem cells to indicate the probable and unwanted changes related to transduction procedure. Materials and Methods In this experimental study, lentiviral vector containing green fluorescent protein (GFP were transduced into unrestricted somatic stem cells and its effect was investigated with using MTT assay, qPCR and immunohistochemistry techniques. For statistical comparison of real time PCR results, REST software (2009, Qiagen was used. Results Obtained results showed lentiviral vector transduction did not have cytotoxic effects on unrestricted somatic stem cells and did not change neuronal differentiation capacity of them as well the expression of some neuronal related genes and preserved them in multilineage situation. Conclusions In conclusion, we suggested that lentiviral vectors could be proper vectors to transfer therapeutic gene into unrestricted somatic stem cells to provide a cellular vehicle for ex vivo gene therapy of nervous system disorders.

  1. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, Hiromasa [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Oki, Yoshinao [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan); Bono, Hidemasa [Database Center for Life Science (DBCLS), Research Organization of Information and Systems (ROIS), Faculty of Engineering Bldg.12 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Kano, Koichiro, E-mail: kkano@brs.nihon-u.ac.jp [Laboratory of Cell and Tissue Biology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510 (Japan)

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  2. Multi-gene epigenetic silencing of tumor suppressor genes in T-cell lymphoma cells; delayed expression of the p16 protein upon reversal of the silencing

    DEFF Research Database (Denmark)

    Nagasawa, T; Zhang, Q; Raghunath, P N

    2006-01-01

    To understand better T-cell lymphomagenesis, we examined promoter CpG methylation and mRNA expression of closely related genes encoding p16, p15, and p14 tumor suppressor genes in cultured malignant T-cells that were derived from cutaneous, adult type, and anaplastic lymphoma kinase (ALK)-express...

  3. Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy

    Directory of Open Access Journals (Sweden)

    Hung Jaclyn Y

    2008-09-01

    Full Text Available Abstract Background Musashi1 (Msi1 is an RNA binding protein with a central role during nervous system development and stem cell maintenance. High levels of Msi1 have been reported in several malignancies including brain tumors thereby associating Msi1 and cancer. Methods We used the human medulloblastoma cell line Daoy as model system in this study to knock down the expression of Msi1 and determine the effects upon soft agar growth and neurophere formation. Quantitative RT-PCR was conducted to evaluate the expression of cell proliferation, differentiation and survival genes in Msi1 depleted Daoy cells. Results We observed that MSI1 expression was elevated in Daoy cells cultured as neurospheres compared to those grown as monolayer. These data indicated that Msi1 might be involved in regulating proliferation in cancer cells. Here we show that shRNA mediated Msi1 depletion in Daoy cells notably impaired their ability to form colonies in soft agar and to grow as neurospheres in culture. Moreover, differential expression of a group of Notch, Hedgehog and Wnt pathway related genes including MYCN, FOS, NOTCH2, SMO, CDKN1A, CCND2, CCND1, and DKK1, was also found in the Msi1 knockdown, demonstrating that Msi1 modulated the expression of a subset of cell proliferation, differentiation and survival genes in Daoy. Conclusion Our data suggested that Msi1 may promote cancer cell proliferation and survival as its loss seems to have a detrimental effect in the maintenance of medulloblastoma cancer cells. In this regard, Msi1 might be a positive regulator of tumor progression and a potential target for therapy.

  4. Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    David T. Ting

    2014-09-01

    Full Text Available Circulating tumor cells (CTCs are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

  5. Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

    OpenAIRE

    1993-01-01

    Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow...

  6. Gene expression analysis uncovers novel hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells.

    Science.gov (United States)

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J Fah; Cho, Michael H; Mancini, John D; Lao, Taotao; Thibault, Derek M; Litonjua, Augusto A; Bakke, Per S; Gulsvik, Amund; Lomas, David A; Beaty, Terri H; Hersh, Craig P; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A; Rennard, Stephen I; Perrella, Mark A; Choi, Augustine M K; Quackenbush, John; Silverman, Edwin K

    2013-05-01

    Hedgehog interacting protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.

  7. Annotation and classification of the bovine T cell receptor delta genes.

    Science.gov (United States)

    Herzig, Carolyn T A; Lefranc, Marie-Paule; Baldwin, Cynthia L

    2010-02-09

    gammadelta T cells differ from alphabeta T cells with regard to the types of antigen with which their T cell receptors interact; gammadelta T cell antigens are not necessarily peptides nor are they presented on MHC. Cattle are considered a "gammadelta T cell high" species indicating they have an increased proportion of gammadelta T cells in circulation relative to that in "gammadelta T cell low" species such as humans and mice. Prior to the onset of the studies described here, there was limited information regarding the genes that code for the T cell receptor delta chains of this gammadelta T cell high species. By annotating the bovine (Bos taurus) genome Btau_3.1 assembly the presence of 56 distinct T cell receptor delta (TRD) variable (V) genes were found, 52 of which belong to the TRDV1 subgroup and were co-mingled with the T cell receptor alpha variable (TRAV) genes. In addition, two genes belonging to the TRDV2 subgroup and single TRDV3 and TRDV4 genes were found. We confirmed the presence of five diversity (D) genes, three junctional (J) genes and a single constant (C) gene and describe the organization of the TRD locus. The TRDV4 gene is found downstream of the C gene and in an inverted orientation of transcription, consistent with its orthologs in humans and mice. cDNA evidence was assessed to validate expression of the variable genes and showed that one to five D genes could be incorporated into a single transcript. Finally, we grouped the bovine and ovine TRDV1 genes into sets based on their relatedness. The bovine genome contains a large and diverse repertoire of TRD genes when compared to the genomes of "gammadelta T cell low" species. This suggests that in cattle gammadelta T cells play a more important role in immune function since they would be predicted to bind a greater variety of antigens.

  8. Inhibitory Effect of Isoflavones on Prostate Cancer Cells and PTEN Gene

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To explore the mechanisms by which genistein and daidzein inhibit the growth of prostate cancer cells. Methods LNCaP and PC-3 cells were exposed to genistein and daidzein and cell viability was determined by MTT assay and cytotoxicity of the drugs by LDH test. Flow cytometry (FCM) was used to assess the cell cycle in LNCaP and PC-3 cells.Reverse transcription-polymerase chain reaction (RT-PCR) was applied to examine the expression of PTEN gene (a tumor suppressor gene), estrogen receptor alpha gene (Erα), estrogen receptor beta gene (Erβ), androgen receptor gene (AR) and vascular endothelial growth factor gene (VEGF). Results The viability of PC-3 and LNCaP cells decreased with increasing concentrations and exposure time of genistein and daidzein. Genistein increased G2/M phase cells in PC-3 cells while decreased S phase cells in LNCaP cells in a dose-dependent manner. Daidzein exerted no influence on the cell cycle of LNCaP and PC-3 cells, but the apoptosis percentage of LNCaP cells was elevated significantly by daidzein. Genistein induced the expression of PTEN gene in PC-3 and LNCaP cells. Daidzein induced the expression of PTEN gene in LNCaP but not in PC-3 cells. The expression of VEGF, Erα and Erβ genes decreased and AR gene was not expressed after incubation with genistein and daidzein in PC-3 cells. In LNCaP cells, the expression of VEGF and AR gene decreased but there was no change in the expression of Erα and Erβ gene after incubation with genistein and daidzein. Conclusion Genistein and daidzein exert a time- and dose-dependent inhibitory effect on PC-3 and LNCaP cells. The down-regulation of ER gene by daidzein influences the growth of PC-3 cells directly. The inhibition of PC-3 cells by genistein and that of LNCaP cells by genistein and daidzein may be via Akt pathway that is repressed by PTEN gene, which subsequently down-regulates the expression of AR and VEGF genes. Our results suggest that the expression of PTEN gene plays a key

  9. Genome-wide gene amplification during differentiation of neural progenitor cells in vitro.

    Science.gov (United States)

    Fischer, Ulrike; Keller, Andreas; Voss, Meike; Backes, Christina; Welter, Cornelius; Meese, Eckart

    2012-01-01

    DNA sequence amplification is a phenomenon that occurs predictably at defined stages during normal development in some organisms. Developmental gene amplification was first described in amphibians during gametogenesis and has not yet been described in humans. To date gene amplification in humans is a hallmark of many tumors. We used array-CGH (comparative genomic hybridization) and FISH (fluorescence in situ hybridization) to discover gene amplifications during in vitro differentiation of human neural progenitor cells. Here we report a complex gene amplification pattern two and five days after induction of differentiation of human neural progenitor cells. We identified several amplified genes in neural progenitor cells that are known to be amplified in malignant tumors. There is also a striking overlap of amplified chromosomal regions between differentiating neural progenitor cells and malignant tumor cells derived from astrocytes. Gene amplifications in normal human cells as physiological process has not been reported yet and may bear resemblance to developmental gene amplifications in amphibians and insects.

  10. Parathyroid hormone-related protein regulates tumor-relevant genes in breast cancer cells.

    NARCIS (Netherlands)

    Dittmer, A.; Vetter, M.; Schunke, D.; Span, P.N.; Sweep, C.G.J.; Thomssen, C.; Dittmer, J.

    2006-01-01

    The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered

  11. Aristaless related homeobox gene, Arx, is implicated in mouse fetal Leydig cell differentiation possibly through expressing in the progenitor cells.

    Directory of Open Access Journals (Sweden)

    Kanako Miyabayashi

    Full Text Available Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage.

  12. Frequent mutations of genes encoding ubiquitin-mediated proteolysis pathway components in clear cell renal cell carcinoma

    DEFF Research Database (Denmark)

    Guo, Guangwu; Gui, Yaoting; Gao, Shengjie;

    2012-01-01

    We sequenced whole exomes of ten clear cell renal cell carcinomas (ccRCCs) and performed a screen of similar to 1,100 genes in 88 additional ccRCCs, from which we discovered 12 previously unidentified genes mutated at elevated frequencies in ccRCC. Notably, we detected frequent mutations in the u......We sequenced whole exomes of ten clear cell renal cell carcinomas (ccRCCs) and performed a screen of similar to 1,100 genes in 88 additional ccRCCs, from which we discovered 12 previously unidentified genes mutated at elevated frequencies in ccRCC. Notably, we detected frequent mutations...

  13. A novel gene delivery system for mammalian cells.

    Science.gov (United States)

    Gibson, Brian; Duffy, Angela M; Gould Fogerite, Susan; Krause-Elsmore, Sara; Lu, Ruying; Shang, Gaofeng; Chen, Zi-Wei; Mannino, Raphael J; Bouchier-Hayes, David J; Harmey, Judith H

    2004-01-01

    Although gene therapy holds great promise for the treatment of both acquired and genetic diseases, its development has been limited by practical considerations. Non-viral efficacy of delivery remains quite poor. We are investigating the feasibility of a novel lipid-based delivery system, cochleates, to deliver transgenes to mammalian cells. Rhodamine-labelled empty cochleates were incubated with two cell-lines (4T1 adenocarcinoma and H36.12 macrophage hybridoma) and primary macrophages in vitro and in vivo. Cochleates containing green fluorescent protein (GFP) expression plasmid were incubated with 4T1 adenocarcinoma cells. Cellular uptake of labelled cochleates or transgene GFP expression were visualised with fluorescence microscopy. 4T1 and H36.12 lines showed 39% and 23.1% uptake of rhodamine-cochleates, respectively. Human monocyte-derived macrophages and mouse peritoneal macrophages had 48+/-5.38% and 51.46+/-15.6% uptake of rhodamine-cochleates in vitro. In vivo 25.69+/-0.127% of peritoneal macrophages were rhodamine-positive after intra-peritoneal injection of rhodamine-cochleates. 19.49+/-10.12% of 4T1 cells expressed GFP. Cochleates may therefore be an effective, non-toxic and non-immunogenic method to introduce transgenes in vitro and in vivo.

  14. Bach2 represses plasma cell gene regulatory network in B cells to promote antibody class switch.

    Science.gov (United States)

    Muto, Akihiko; Ochiai, Kyoko; Kimura, Yoshitaka; Itoh-Nakadai, Ari; Calame, Kathryn L; Ikebe, Dai; Tashiro, Satoshi; Igarashi, Kazuhiko

    2010-12-01

    Two transcription factors, Pax5 and Blimp-1, form a gene regulatory network (GRN) with a double-negative loop, which defines either B-cell (Pax5 high) or plasma cell (Blimp-1 high) status as a binary switch. However, it is unclear how this B-cell GRN registers class switch DNA recombination (CSR), an event that takes place before the terminal differentiation to plasma cells. In the absence of Bach2 encoding a transcription factor required for CSR, mouse splenic B cells more frequently and rapidly expressed Blimp-1 and differentiated to IgM plasma cells as compared with wild-type cells. Genetic loss of Blimp-1 in Bach2(-/-) B cells was sufficient to restore CSR. These data with mathematical modelling of the GRN indicate that Bach2 achieves a time delay in Blimp-1 induction, which inhibits plasma cell differentiation and promotes CSR (Delay-Driven Diversity model for CSR). Reduction in mature B-cell numbers in Bach2(-/-) mice was not rescued by Blimp-1 ablation, indicating that Bach2 regulates B-cell differentiation and function through Blimp-1-dependent and -independent GRNs.

  15. Inferring single-cell gene expression mechanisms using stochastic simulation

    Science.gov (United States)

    Daigle, Bernie J.; Soltani, Mohammad; Petzold, Linda R.; Singh, Abhyudai

    2015-01-01

    Motivation: Stochastic promoter switching between transcriptionally active (ON) and inactive (OFF) states is a major source of noise in gene expression. It is often implicitly assumed that transitions between promoter states are memoryless, i.e. promoters spend an exponentially distributed time interval in each of the two states. However, increasing evidence suggests that promoter ON/OFF times can be non-exponential, hinting at more complex transcriptional regulatory architectures. Given the essential role of gene expression in all cellular functions, efficient computational techniques for characterizing promoter architectures are critically needed. Results: We have developed a novel model reduction for promoters with arbitrary numbers of ON and OFF states, allowing us to approximate complex promoter switching behavior with Weibull-distributed ON/OFF times. Using this model reduction, we created bursty Monte Carlo expectation-maximization with modified cross-entropy method (‘bursty MCEM2’), an efficient parameter estimation and model selection technique for inferring the number and configuration of promoter states from single-cell gene expression data. Application of bursty MCEM2 to data from the endogenous mouse glutaminase promoter reveals nearly deterministic promoter OFF times, consistent with a multi-step activation mechanism consisting of 10 or more inactive states. Our novel approach to modeling promoter fluctuations together with bursty MCEM2 provides powerful tools for characterizing transcriptional bursting across genes under different environmental conditions. Availability and implementation: R source code implementing bursty MCEM2 is available upon request. Contact: absingh@udel.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25573914

  16. CD133-targeted gene transfer into long-term repopulating hematopoietic stem cells.

    Science.gov (United States)

    Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwäble, Joachim; Kaufmann, Kerstin B; Müller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J; Grez, Manuel

    2015-01-01

    Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

  17. Association Mapping of Cell Wall Synthesis Regulatory Genes and Cell Wall Quality in Switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Bartley, Laura [Univ. of Oklahoma, Norman, OK (United States). Dept. of Microbiology and Plant Biology; Wu, Y. [Oklahoma State Univ., Stillwater, OK (United States); Zhu, L. [Oklahoma State Univ., Stillwater, OK (United States); Brummer, E. C. [Noble Foundation, Ardmore, OK (United States); Saha, M. [Noble Foundation, Ardmore, OK (United States)

    2016-05-31

    Inefficient conversion of biomass to biofuels is one of the main barriers for biofuel production from such materials. Approximately half of polysaccharides in biomass remain unused by typical biochemical conversion methods. Conversion efficiency is influenced by the composition and structure of cell walls of biomass. Grasses such as wheat, maize, and rice, as well as dedicated perennial bioenergy crops, like switchgrass, make up ~55% of biomass that can be produced in the United States. Grass cell walls have a different composition and patterning compared with dicotyledonous plants, including the well-studied model plant, Arabidopsis. This project identified genetic determinants of cell wall composition in grasses using both naturally occurring genetic variation of switchgrass and gene network reconstruction and functional assays in rice. In addition, the project linked functional data in rice and other species to switchgrass improvement efforts through curation of the most abundant class of regulators in the switchgrass genome. Characterizing natural diversity of switchgrass for variation in cell wall composition and properties, also known as quality, provides an unbiased avenue for identifying biologically viable diversity in switchgrass cell walls. To characterizing natural diversity, this project generated cell wall composition and enzymatic deconstruction data for ~450 genotypes of the Switchgrass Southern Association Collection (SSAC), a diverse collection composed of 36 switchgrass accessions from the southern U.S. distribution of switchgrass. Comparing these data with other measures of cell wall quality for the same samples demonstrated the complementary nature of the diverse characterization platforms now being used for biomass characterization. Association of the composition data with ~3.2K single nucleotide variant markers identified six significant single nucleotide variant markers co-associated with digestibility and another compositional trait. These

  18. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    Science.gov (United States)

    Gambhir; Sanjiv , Pritha; Ray

    2009-04-28

    Novel double and triple fusion reporter gene constructs harboring distinct imageable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  19. Analysis of target genes induced by IL-13 cytotoxin in human glioblastoma cells.

    Science.gov (United States)

    Han, Jing; Yang, Liming; Puri, Raj K

    2005-03-01

    IL-13 cytotoxin comprised of IL-13 and a mutated form of Pseudomonas exotoxin (fusion protein termed IL-13-PE38QQR) has been shown to inhibit protein synthesis leading to necrotic and apoptotic cell death in glioblastoma cells that express high levels of interleukin-13 receptors (IL-13R). To identify target genes of cell death and other cellular genes with IL-13 receptors in glioblastoma cells, we utilized the cDNA microarrays to analyze global gene expression profiles after IL-13 cytotoxin and IL-13 treatment. IL-13 cytotoxin mediated cytotoxicity to U251 cells in a dose-dependent manner. Hierarchical cluster analysis of differentially expressed genes in U251 glioma cells at different time points after IL-13 cytotoxin treatment showed three major groups, each representing a specific expression pattern. Randomly selected differentially expressed genes from each group were confirmed by RT-PCR analysis. Most down-regulated genes belong to cell adhesion, motility, angiogenesis, DNA repair, and metabolic pathways. While up-regulated genes belong to cell cycle arrest, apoptosis, signaling and various metabolic pathways. Unexpectedly, at early time points, both IL-13 and IL-13 cytotoxin induced several genes belonging to different pathways most notably IL-8, DIO2, END1, and ALDH1A3 indicating that these genes are early response genes and their products may be associated with IL-13R. In addition, IL-13 cytotoxin induced IL-13Ralpha2 mRNA expression during the treatment in glioma cells. Our results indicate that novel cellular genes are involved with IL-13 receptors and that IL-13 cytotoxin induced cell death involves various target genes in human glioblastoma cells. On going studies will determine the role of associated genes and their products in the IL-13R functions in glioma cells.

  20. The association of killer cell immunoglobulin like receptor gene polylmorphism with cytomegalovirus infection after hematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    吴小津

    2013-01-01

    Objective To explore the influence of the killer cell immunoglobulin like receptor(KIR)gene polymorphism on cytomegalovirus(CMV)infection and pathogenesis after hematopoietic stem cell transplantation(HSCT)

  1. Silencing of Taxol-Sensitizer Genes in Cancer Cells: Lack of Sensitization Effects

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shang-Lang [Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chao, Chuck C.-K., E-mail: cckchao@mail.cgu.edu.tw [Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Department of Medical Research and Development, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan (China)

    2015-06-16

    A previous genome-wide screening analysis identified a panel of genes that sensitize the human non-small-cell lung carcinoma cell line NCI-H1155 to taxol. However, whether the identified genes sensitize other cancer cells to taxol has not been examined. Here, we silenced the taxol-sensitizer genes identified (acrbp, atp6v0d2, fgd4, hs6st2, psma6, and tubgcp2) in nine other cancer cell types (including lung, cervical, ovarian, and hepatocellular carcinoma cell lines) that showed reduced cell viability in the presence of a sub-lethal concentration of taxol. Surprisingly, none of the genes studied increased sensitivity to taxol in the tested panel of cell lines. As observed in H1155 cells, SKOV3 cells displayed induction of five of the six genes studied in response to a cell killing dose of taxol. The other cell types were much less responsive to taxol. Notably, four of the five inducible taxol-sensitizer genes tested (acrbp, atp6v0d2, psma6, and tubgcp2) were upregulated in a taxol-resistant ovarian cancer cell line. These results indicate that the previously identified taxol-sensitizer loci are not conserved genetic targets involved in inhibiting cell proliferation in response to taxol. Our findings also suggest that regulation of taxol-sensitizer genes by taxol may be critical for acquired cell resistance to the drug.

  2. Comparative analysis of gene expression in normal and cancer human prostate cell lines

    Directory of Open Access Journals (Sweden)

    E. E. Rosenberg

    2014-04-01

    Full Text Available Prostate cancer is one of the main causes of mortality in men with malignant tumors. The urgent problem was a search for biomarkers of prostate cancer, which would allow distinguishing between aggressive metastatic and latent tumors. The aim of this work was to search for differentially expressed genes in normal epithelial cells PNT2 and prostate cancer cell lines LNCaP, DU145 and PC3, produced from tumors with different aggressiveness and metas­tatic ability. Such genes might be used to create a panel of prognostic markers for aggressiveness and metastasis. Relative gene expression of 65 cancer-related genes was determined by the quantitative polymerase chain reaction (Q-PCR. Expression of 29 genes was changed in LNCaP cells, 20 genes in DU145 and 16 genes in PC3 cell lines, compared with normal line PNT2. The obtained data make it possible to conclude that the epithelial-mesenchymal cell transition took place, which involved the loss of epithelial markers, reduced cell adhesion and increased migration. We have also found few differentially expressed genes among 3 prostate cancer cell lines. We have found that genes, involved in cell adhesion (CDH1, invasiveness and metastasis (IL8, CXCL2 and cell cycle control (P16, CCNE1 underwent most changes. These genes might be used for diagnosis and prognosis of invasive metastatic prostate tumors.

  3. Gene set enrichment analysis and ingenuity pathway analysis of metastatic clear cell renal cell carcinoma cell line.

    Science.gov (United States)

    Khan, Mohammed I; Dębski, Konrad J; Dabrowski, Michał; Czarnecka, Anna M; Szczylik, Cezary

    2016-08-01

    In recent years, genome-wide RNA expression analysis has become a routine tool that offers a great opportunity to study and understand the key role of genes that contribute to carcinogenesis. Various microarray platforms and statistical approaches can be used to identify genes that might serve as prognostic biomarkers and be developed as antitumor therapies in the future. Metastatic renal cell carcinoma (mRCC) is a serious, life-threatening disease, and there are few treatment options for patients. In this study, we performed one-color microarray gene expression (4×44K) analysis of the mRCC cell line Caki-1 and the healthy kidney cell line ASE-5063. A total of 1,921 genes were differentially expressed in the Caki-1 cell line (1,023 upregulated and 898 downregulated). Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) approaches were used to analyze the differential-expression data. The objective of this research was to identify complex biological changes that occur during metastatic development using Caki-1 as a model mRCC cell line. Our data suggest that there are multiple deregulated pathways associated with metastatic clear cell renal cell carcinoma (mccRCC), including integrin-linked kinase (ILK) signaling, leukocyte extravasation signaling, IGF-I signaling, CXCR4 signaling, and phosphoinositol 3-kinase/AKT/mammalian target of rapamycin signaling. The IPA upstream analysis predicted top transcriptional regulators that are either activated or inhibited, such as estrogen receptors, TP53, KDM5B, SPDEF, and CDKN1A. The GSEA approach was used to further confirm enriched pathway data following IPA.

  4. The human desmin promoter drives robust gene expression for skeletal muscle stem cell-mediated gene therapy.

    Science.gov (United States)

    Jonuschies, Jacqueline; Antoniou, Michael; Waddington, Simon; Boldrin, Luisa; Muntoni, Francesco; Thrasher, Adrian; Morgan, Jennifer

    2014-01-01

    Lentiviral vectors (LVs) represent suitable candidates to mediate gene therapy for muscular dystrophies as they infect dividing and non-dividing cells and integrate their genetic material into the host genome, thereby theoretically mediating longterm expression. We evaluated the ability of LVs where a GFP reporter gene was under the control of five different promoters, to transduce and mediate expression in myogenic and non-myogenic cells in vitro and in skeletal muscle fibres and stem (satellite) cells in vivo. We further analysed lentivirally-transduced satellite cell-derived myoblasts following their transplantation into dystrophic, immunodeficient mouse muscles. The spleen focus-forming virus promoter mediated the highest gene expression in all cell types; the CBX3-HNRPA2B1 ubiquitously-acting chromatin opening element (UCOE) promoter was also active in all cells, whereas the human desmin promoter in isolation or fused with UCOE had lower activity in non-muscle cells. Surprisingly, the human skeletal muscle actin promoter was also active in immune cells. The human desmin promoter mediated robust, persistent reporter gene expression in myogenic cells in vitro, and satellite cells and muscle fibres in vivo. The human desmin promoter combined with UCOE did not significantly increase transgene expression. Therefore, our data indicate that the desmin promoter is suitable for the development of therapeutic purposes.

  5. 75 FR 54351 - Cell and Gene Therapy Clinical Trials in Pediatric Populations; Public Workshop

    Science.gov (United States)

    2010-09-07

    ... HUMAN SERVICES Food and Drug Administration Cell and Gene Therapy Clinical Trials in Pediatric... public workshop entitled ``Cell and Gene Therapy Clinical Trials in Pediatric Populations.'' The purpose... therapy clinical researchers, and other stakeholders regarding best practices related to cell and...

  6. EXPRESSION OF T CELL RECEPTOR Vα GENE FAMILIES IN INTRATHYROIDAL T CELLS OF CHINESE PATIENTS WITH GRAVES' DISEASE

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. Patients with Graves' disease (GD) have marked lymphocytic infiltration in their thyroid glands. We examined the gene for the variable regions of the α-chain of the Chinese T-cell receptor( Vα gene) in intrathyroidal Tcells to determine the role of T cells in the pathogenesis of GD and offer potential for the development of immunothera-peutic remedies for GD. Methods. We used the reverse transcription and polymerase chain reaction(RT-PCR) to amplify complementary DNA(cDNA) for the 18 known families of the Vα gene in intrathyroidal T cells from 5 patients with Graves' disease.The findings were compared with the results of peripheral blood T cells in the same patients as well as those in normalsubjects. Results. We found that marked restriction in the expression of T cell receptor Vα genes by T cells from the thyroidtissue of Chinese patients with GD(P < 0.001). An average of only 4.6 ± 1.52 of the 18 Vα genes were expressed insuch samples, as compared with 10.4 ± 2.30Vα genes expressed in peripheral blood T cells from the same patients.The pattem of expressed Vα genes differed from patient to patient with no clear predominance. Condusions. Expression of intrathyroidal T cell receptor Vα genes in GD is highly restricted suggesting the prima-cy of T cells in causing the disorders.

  7. Effects of navelbine and docetaxel on gene expression in lung cancer cell strains

    Institute of Scientific and Technical Information of China (English)

    Li CAI; Hai-ying DONG; Guang-jie SUI

    2005-01-01

    Aim: To search genes sensitivity to the anti-cancer drugs navelbine (NVB) and docetaxel (DOC) in small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cell strains. Methods: The sensitivity of 4 strains of SCLC and 6 strains of NSCLC to NVB and DOC was evaluated using the MTT assay. The expression of 1291 sensitive-related genes to the anti-cancer drugs in 10 lung cancer cell strains was measured using cDNA macroarrays and the relationship was analyzed.Results: In total, there were 56 (r≥0.4) genes sensitive to NVB and DOC. For NVB: 36 genes were sensitive to NVB, 20 co-expressed genes between the SCLC+NSCLC set and the NSCLC set; 27 expressed genes and 7 specially expressed genes in the SCLC+NSCLC set; and 29 expressed genes and 9 specially expressed genes in the NSCLC set. For DOC, 50 genes were sensitive to DOC, 12co-expressed genes between the SCLC+NSCLC set and the NSCLC set; 24expressed genes and 12 specially expressed genes in the SCLC+NSCLC set; and 38 expressed genes and 26 specially expressed genes in the NSCLC set. The genes sensitive to NVB and DOC in lung-cancer cell stains were mainly divided into the following 4 categories: signal transduction molecules, cell factors, transcription factors and metabolism-related enzymes and inhibitors. Conclusions:There were obvious differences in genes related to NVB and DOC between SCLC and NSCLC cell strains, but the same as categories of function.

  8. An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells.

    Science.gov (United States)

    Carter, Mark G; Stagg, Carole A; Falco, Geppino; Yoshikawa, Toshiyuki; Bassey, Uwem C; Aiba, Kazuhiro; Sharova, Lioudmila V; Shaik, Nabeebi; Ko, Minoru S H

    2008-02-01

    We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.

  9. Identification of Suitable Reference Genes for Peripheral Blood Mononuclear Cell Subset Studies in Multiple Sclerosis

    DEFF Research Database (Denmark)

    Oturai, D B; Søndergaard, H B; Börnsen, L;

    2016-01-01

    Quantitative real-time PCR (qPCR) involves the need of a proper standard for normalizing the gene expression data. Different studies have shown the validity of reference genes to vary greatly depending on tissue, cell subsets and experimental context. This study aimed at the identification...... of suitable reference genes for qPCR studies using different peripheral blood cell subsets (whole blood (WB) cells, peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD4(+) T cells, CD8(+) T cells, NK cells, monocytes, B cells and dendritic cells) from healthy controls (HC), patients with relapsing...... stable combination for analyses of cell subsets between HC and RRMS patients, while the combination of UBC and YWHAZ was superior for analysis of cell subsets between HC, RRMS and RRMS-IFN-β groups. GAPDH was generally unsuitable for blood cell subset studies in multiple sclerosis. In conclusion, we...

  10. Circadian Clock Genes Modulate Human Bone Marrow Mesenchymal Stem Cell Differentiation, Migration and Cell Cycle.

    Science.gov (United States)

    Boucher, Helene; Vanneaux, Valerie; Domet, Thomas; Parouchev, Alexandre; Larghero, Jerome

    2016-01-01

    Many of the components that regulate the circadian clock have been identified in organisms and humans. The influence of circadian rhythm (CR) on the regulation of stem cells biology began to be evaluated. However, little is known on the role of CR on human mesenchymal stem cell (hMSCs) properties. The objective of this study was to investigate the influence of CR on the differentiation capacities of bone marrow hMSCs, as well as the regulation of cell cycle and migration capabilities. To that, we used both a chemical approach with a GSK-3β specific inhibitor (2'E,3'Z-6-bromoindirubin-3'-oxime, BIO) and a knockdown of CLOCK and PER2, two of the main genes involved in CR regulation. In these experimental conditions, a dramatic inhibition of adipocyte differentiation was observed, while osteoblastic differentiation capacities were not modified. In addition, cell migration was decreased in PER2-/- cells. Lastly, downregulation of circadian clock genes induced a modification of the hMSCs cell cycle phase distribution, which was shown to be related to a change of the cyclin expression profile. Taken together, these data showed that CR plays a role in the regulation of hMSCs differentiation and division, and likely represent key factor in maintaining hMSCs properties.

  11. Bacterial toxin-antitoxin gene system as containment control in yeast cells

    DEFF Research Database (Denmark)

    Kristoffersen, P.; Jensen, G. B.; Gerdes, K.;

    2000-01-01

    The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae, Expression of the relE gene was highly toxic to yeast cells. However, expression...... of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae, Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial...

  12. Gene delivery to mice spermatogenic stem cells by EffecteneTM reagent

    Institute of Scientific and Technical Information of China (English)

    陈晓光; 王宁; 姚纪花; 陈浩明; 沈琦; 薛京伦

    2004-01-01

    @@ Spermatogenic (stem) cells, or spermatogonial stem cells, are the only cell type in postnatal mammals, which have the capability to self-renew and to contribute geneticinformation to the next generation. The manipulation of spermatogenic cells and the modification of their genomes have great significance for the treatment of male sterility,for gene therapy via germ cells, as well as for building transgenic animal models. 1 In this assay, we analyzed the efficiency of EffecteneTM reagent-mediated gene transfection into spermatogenic cells. The effect of transplants with different time schedules on transfection efficiency and on gene expression was also investigated.

  13. Cell-Specific Promoters Enable Lipid-Based Nanoparticles to Deliver Genes to Specific Cells of the Retina In Vivo.

    Science.gov (United States)

    Wang, Yuhong; Rajala, Ammaji; Cao, Binrui; Ranjo-Bishop, Michelle; Agbaga, Martin-Paul; Mao, Chuanbin; Rajala, Raju V S

    2016-01-01

    Non-viral vectors, such as lipid-based nanoparticles (liposome-protamine-DNA complex [LPD]), could be used to deliver a functional gene to the retina to correct visual function and treat blindness. However, one of the limitations of LPD is the lack of cell specificity, as the retina is composed of seven types of cells. If the same gene is expressed in multiple cell types or is absent from one desired cell type, LPD-mediated gene delivery to every cell may have off-target effects. To circumvent this problem, we have tested LPD-mediated gene delivery using various generalized, modified, and retinal cell-specific promoters. We achieved retinal pigment epithelium cell specificity with vitelliform macular dystrophy (VMD2), rod cell specificity with mouse rhodopsin, cone cell specificity with red/green opsin, and ganglion cell specificity with thymocyte antigen promoters. Here we show for the first time that cell-specific promoters enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo. This work will inspire investigators in the field of lipid nanotechnology to couple cell-specific promoters to drive expression in a cell- and tissue-specific manner.

  14. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

    OpenAIRE

    Klein, U.; Klein, G.; Ehlin-Henriksson, B.; Rajewsky, K.; Küppers, R.

    1995-01-01

    BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells a...

  15. Gene targeting in a HUES line of human embryonic stem cells via electroporation.

    Science.gov (United States)

    Ruby, Katherine M; Zheng, Binhai

    2009-07-01

    Genetic modification is critical for achieving the full potential of human embryonic stem (ES) cells as a tool for therapeutic development and for basic research. Targeted modifications in human ES cells have met with limited success because of the unique culture conditions for many human ES cell lines. The HUES lines of human ES cells were developed for ease of manipulation and are gaining increased utility in stem cell research. We tested conditions for gene targeting via electroporation in the HUES-9 human ES cell line and demonstrate here successful gene targeting at the gene encoding Fezf2 (also known as Fezl), a transcription factor involved in corticospinal neuron development. With a targeting strategy involving positive and negative selection that is applicable to all genes, we observed a gene targeting frequency of approximately 1.5% for Fezf2, a gene not expressed in human ES cells. We found that conditions developed for gene targeting in mouse ES cells can be readily adapted to HUES cells with few key modifications. HUES-9 cells exhibit an intrinsically high efficiency of clonal expansion and sustain electroporation-based gene targeting procedures without any significant loss of pluripotency marker expression or karyotypic stability. Thus, human ES cell lines adapted for enzymatic passage and efficient clonal expansion can be highly amenable to genetic modifications, which will facilitate their application in basic science and clinical development.

  16. 3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Hatta, Mitsutoki, E-mail: hatta@college.fdcnet.ac.jp [Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka (Japan); Naganuma, Kaori [Department of Oral and Maxillofacial Surgery, Fukuoka Dental College, Fukuoka (Japan); Kato, Kenichi; Yamazaki, Jun [Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka (Japan)

    2015-12-04

    In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial–mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell–cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. - Highlights: • DZNep reduced PcG proteins and associated histone modifications in OSCC cells. • DZNep enhanced cell–cell adhesion indicative of epithelial phenotype in OSCC cells. • DZNep suppressed the aggressive phenotype-related gene expression in OSCC cells. • DZNep activated the gene expression of epithelial markers in OSCC cells.

  17. Evidence for the multicentric origin of the sickle cell hemoglobin gene in Africa.

    OpenAIRE

    Pagnier, J.; Mears, J G; Dunda-Belkhodja, O; Schaefer-Rego, K E; Beldjord, C; Nagel, R L; Labie, D

    1984-01-01

    Previous studies of the Hpa I cleavage site-sickle cell hemoglobin gene linkage in various African populations suggested that the sickle gene arose independently more than once. In the present study we have performed restriction endonuclease haplotype analysis for the beta-globin-like gene cluster from four separate geographic areas in Africa, all of which possess the sickle gene. In Benin (Central West Africa) and Algeria (Arab North Africa) all chromosomes carrying the sickle gene possess a...

  18. Meta-analysis of differentiating mouse embryonic stem cell gene expression kinetics reveals early change of a small gene set.

    Directory of Open Access Journals (Sweden)

    Clive H Glover

    2006-11-01

    Full Text Available Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42 showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.

  19. Effects of emodin on gene expression profile in small cell lung cancer NCI-H446 cells

    Institute of Scientific and Technical Information of China (English)

    FU Zhong-yan; HAN Jin-xiang; HUANG Hai-yan

    2007-01-01

    Background The treatment of patients with small cell lung cancer (SCLC) is based on chemotherapy. However, the treatment is limited by the development of drug resistance. Emodin has been shown to exhibit an anti-cancer effect. But the molecular mechanism remains unclear. This study was conducted to investigate the effect of emodin on the gene expression profile changes in SCLC NCI-H446 cells.Methods NCI-H446 cells were treated with emodin and cell viability was determined by MTT assay. Cell apoptosis was determined by both flow cytometry and caspase-3 activity assay. The effect of emodin on the gene expression profile of NCI-H446 cells was analyzed using cDNA microarray. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to validate the microarray results.Results Emodin suppressed viability, induced apoptosis and changed cell cycle of NCI-H446 cells. Among the 1262 genes, 10 genes were up-regulated and 8 genes were down-regulated more than 2 folds in NCI-H446 cells when compared with the control cells after treatment with emodin for 12 hours, while 12 genes were up-regulated and 24 genes were down-regulated after treatment with emodin for 24 hours. These genes were involved in metabolism, signal transduction, transcription regulation, cytoskeleton organization, immune response, transport, protein synthesis, cell cycle control, cell adhesion and RNA processing. The RT-PCR results were consistent with those obtained by the microarray.Conclusions Emodin affects the expression of genes involved in various cellular functions and plays important roles in cell apoptosis, tumor metastasis and chemotherapy-resistance, which suggests emodin might become an effective chemopreventive or chemotherapeutic agent for SCLC.

  20. Genes Required for Bacillus anthracis Secondary Cell Wall Polysaccharide Synthesis

    Science.gov (United States)

    Oh, So-Young; Lunderberg, J. Mark; Chateau, Alice; Schneewind, Olaf

    2016-01-01

    ABSTRACT The secondary cell wall polysaccharide (SCWP) is thought to be essential for vegetative growth and surface (S)-layer assembly in Bacillus anthracis; however, the genetic determinants for the assembly of its trisaccharide repeat structure are not known. Here, we report that WpaA (BAS0847) and WpaB (BAS5274) share features with membrane proteins involved in the assembly of O-antigen lipopolysaccharide in Gram-negative bacteria and propose that WpaA and WpaB contribute to the assembly of the SCWP in B. anthracis. Vegetative forms of the B. anthracis wpaA mutant displayed increased lengths of cell chains, a cell separation defect that was attributed to mislocalization of the S-layer-associated murein hydrolases BslO, BslS, and BslT. The wpaB mutant was defective in vegetative replication during early logarithmic growth and formed smaller colonies. Deletion of both genes, wpaA and wpaB, did not yield viable bacilli, and when depleted of both wpaA and wpaB, B. anthracis could not maintain cell shape, support vegetative growth, or assemble SCWP. We propose that WpaA and WpaB fulfill overlapping glycosyltransferase functions of either polymerizing repeat units or transferring SCWP polymers to linkage units prior to LCP-mediated anchoring of the polysaccharide to peptidoglycan. IMPORTANCE The secondary cell wall polysaccharide (SCWP) is essential for Bacillus anthracis growth, cell shape, and division. SCWP is comprised of trisaccharide repeats (→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→) with α-Gal and β-Gal substitutions; however, the genetic determinants and enzymes for SCWP synthesis are not known. Here, we identify WpaA and WpaB and report that depletion of these factors affects vegetative growth, cell shape, and S-layer assembly. We hypothesize that WpaA and WpaB are involved in the assembly of SCWP prior to transfer of this polymer onto peptidoglycan. PMID:27795328

  1. Microarray Analysis on Gene Regulation by Estrogen, Progesterone and Tamoxifen in Human Endometrial Stromal Cells

    Directory of Open Access Journals (Sweden)

    Chun-E Ren

    2015-03-01

    Full Text Available Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies.

  2. Microarray Analysis on Gene Regulation by Estrogen, Progesterone and Tamoxifen in Human Endometrial Stromal Cells

    Science.gov (United States)

    Ren, Chun-E; Zhu, Xueqiong; Li, Jinping; Lyle, Christian; Dowdy, Sean; Podratz, Karl C.; Byck, David; Chen, Hai-Bin; Jiang, Shi-Wen

    2015-01-01

    Epithelial stromal cells represent a major cellular component of human uterine endometrium that is subject to tight hormonal regulation. Through cell-cell contacts and/or paracrine mechanisms, stromal cells play a significant role in the malignant transformation of epithelial cells. We isolated stromal cells from normal human endometrium and investigated the morphological and transcriptional changes induced by estrogen, progesterone and tamoxifen. We demonstrated that stromal cells express appreciable levels of estrogen and progesterone receptors and undergo different morphological changes upon hormonal stimulation. Microarray analysis indicated that both estrogen and progesterone induced dramatic alterations in a variety of genes associated with cell structure, transcription, cell cycle, and signaling. However, divergent patterns of changes, and in some genes opposite effects, were observed for the two hormones. A large number of genes are identified as novel targets for hormonal regulation. These hormone-responsive genes may be involved in normal uterine function and the development of endometrial malignancies. PMID:25782154

  3. Forced expression of the Oct-4 gene influences differentiation of embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    By transfecting an Oct-4 expression plasmid into embryonic stem cells (ES cells), the ES-O cell line was constructed, which sustained the expression of Oct-4 gene when induced by retinoic acid. Forced expression of Oct-4 gene could not sustain the stem property of ES-O cells without the differentiation inhibiting factor LIF, but if LIF exists,forced expression of Oct-4 gene could enhance the ability to sustain the undifferentiation state and inhibit cell differentiation induced by retinoic acid. It was indicated that Oct-4 must cooperate with LIF to sustain the undifferentiation state of ES cells. During the cell differentiation, ES-O cells tend to differentiate into neural cells, suggesting that forced expression of Oct-4 gene may be in relation with the differentiation of neuroderm.

  4. Filaggrin Gene Mutations and Risk of Basal Cell Carcinoma

    DEFF Research Database (Denmark)

    Kaae, Jesper Rabølle; Thyssen, J P; Johansen, J D

    2013-01-01

    Basal cell carcinoma (BCC) is prevalent in lightly-pigmented Europeans. While ultraviolet (UV) radiation is an important risk factor, genetic predispositions to BCC have also been identified (1) . Atopic dermatitis (AD), a condition with a heritability that reaches 71-84%, might increase the risk...... of BCC (2) . Loss-of-function mutations in the filaggrin gene (FLG) are observed in approximately 10% of Northern Europeans and are strongly associated with AD (3) . FLG mutations lead to reduced epidermal filaggrin protein and metabolite levels, including the chromophore trans-urocanic acid (UCA) (4......) . Mice with knockdown of filaggrin, or lack of functional histidase, show decreased epidermal trans-UCA levels and increased UVB-induced skin damage (5) . FLG mutation carriers also have 10% increased serum vitamin D levels suggesting increased penetration of UVB (6) . We evaluated the prevalence of FLG...

  5. Evaluation of the free-radical-scavenging activity of diclofenac acid on the free-radical-induced haemolysis of human erythrocytes.

    Science.gov (United States)

    Tang, You-Zhi; Liu, Zai-Qun

    2006-05-01

    Free-radical-induced peroxidation in-vivo is regarded as the aetiology of some diseases and free-radical-scavenging drugs, also called antioxidants (AH), have been widely used to overcome oxidative stress. An in-vitro experimental method, 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced haemolysis of human erythrocytes can be applied to assess the free-radical-scavenging activity of a drug. The major objectives of this work were focused on three aspects. Firstly, introduction of the chemical kinetic deduction of free-radical-initiating reaction to AAPH-induced haemolysis of human erythrocytes, by which the number of free radicals trapped by an antioxidant, n, can be obtained after finding the quantitative relationship between the inhibition period (t(inh)) and the concentration of the antioxidant, t(inh) = (n/Ri) [AH]. Ri, the free-radical-initiating rate, was initially confirmed by using alpha-tocopherol (VE) whose n was taken as 2. Secondly, the free-radical-scavenging activity of diclofenac acid (DaH) and its sodium salt (DaNaH) was assessed. It has been found that DaH and DaNaH protect human erythrocytes against AAPH-induced haemolysis dose-dependently. In particular, the n values of DaH and DaNaH (4.96 and 3.60) were much higher than some traditional antioxidants, such as 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a water-soluble structural analogue of VE, n = 0.30) and L-ascorbic acid (VC, n = 0.25), and L-ascorbyl-6-laurate (VC-12, a lipophilic structural analogue of VC, n = 1.11). Moreover, the free-radical-scavenging activity of lipophilic antioxidants is higher than the corresponding water-soluble species. Thirdly, the free-radical-scavenging activity of mixed antioxidants, VE + DaH, VC-12 + DaH, Trolox + DaNaH and VC + DaNaH, was revealed. The n value of VC, VC-12, VE and Trolox increase in the case of mixed usage with DaH and DaNaH, implying that diclofenac acid can repair the radical of these antioxidants. Thus, a mutual

  6. Time- and dose-dependent effects of curcumin on gene expression in human colon cancer cells

    Directory of Open Access Journals (Sweden)

    van Erk Marjan J

    2004-05-01

    Full Text Available Abstract Background Curcumin is a spice and a coloring food compound with a promising role in colon cancer prevention. Curcumin protects against development of colon tumors in rats treated with a colon carcinogen, in colon cancer cells curcumin can inhibit cell proliferation and induce apoptosis, it is an anti-oxidant and it can act as an anti-inflammatory agent. The aim of this study was to elucidate mechanisms and effect of curcumin in colon cancer cells using gene expression profiling. Methods Gene expression changes in response to curcumin exposure were studied in two human colon cancer cell lines, using cDNA microarrays with four thousand human genes. HT29 cells were exposed to two different concentrations of curcumin and gene expression changes were followed in time (3, 6, 12, 24 and 48 hours. Gene expression changes after short-term exposure (3 or 6 hours to curcumin were also studied in a second cell type, Caco-2 cells. Results Gene expression changes (>1.5-fold were found at all time points. HT29 cells were more sensitive to curcumin than Caco-2 cells. Early response genes were involved in cell cycle, signal transduction, DNA repair, gene transcription, cell adhesion and xenobiotic metabolism. In HT29 cells curcumin modulated a number of cell cycle genes of which several have a role in transition through the G2/M phase. This corresponded to a cell cycle arrest in the G2/M phase as was observed by flow cytometry. Functional groups with a similar expression profile included genes involved in phase-II metabolism that were induced by curcumin after 12 and 24 hours. Expression of some cytochrome P450 genes was downregulated by curcumin in HT29 and Caco-2 cells. In addition, curcumin affected expression of metallothionein genes, tubulin genes, p53 and other genes involved in colon carcinogenesis. Conclusions This study has extended knowledge on pathways or processes already reported to be affected by curcumin (cell cycle arrest, phase

  7. Reconstruction of gene regulatory modules in cancer cell cycle by multi-source data integration.

    Directory of Open Access Journals (Sweden)

    Yuji Zhang

    Full Text Available BACKGROUND: Precise regulation of the cell cycle is crucial to the growth and development of all organisms. Understanding the regulatory mechanism of the cell cycle is crucial to unraveling many complicated diseases, most notably cancer. Multiple sources of biological data are available to study the dynamic interactions among many genes that are related to the cancer cell cycle. Integrating these informative and complementary data sources can help to infer a mutually consistent gene transcriptional regulatory network with strong similarity to the underlying gene regulatory relationships in cancer cells. RESULTS AND PRINCIPAL FINDINGS: We propose an integrative framework that infers gene regulatory modules from the cell cycle of cancer cells by incorporating multiple sources of biological data, including gene expression profiles, gene ontology, and molecular interaction. Among 846 human genes with putative roles in cell cycle regulation, we identified 46 transcription factors and 39 gene ontology groups. We reconstructed regulatory modules to infer the underlying regulatory relationships. Four regulatory network motifs were identified from the interaction network. The relationship between each transcription factor and predicted target gene groups was examined by training a recurrent neural network whose topology mimics the network motif(s to which the transcription factor was assigned. Inferred network motifs related to eight well-known cell cycle genes were confirmed by gene set enrichment analysis, binding site enrichment analysis, and comparison with previously published experimental results. CONCLUSIONS: We established a robust method that can accurately infer underlying relationships between a given transcription factor and its downstream target genes by integrating different layers of biological data. Our method could also be beneficial to biologists for predicting the components of regulatory modules in which any candidate gene is involved

  8. Mesenchymal stromal cells retrovirally transduced with prodrug-converting genes are suitable vehicles for cancer gene therapy.

    Science.gov (United States)

    Ďuriniková, E; Kučerová, L; Matúšková, M

    2014-01-01

    Mesenchymal stem/stromal cells (MSC) possess a set of several fairly unique properties which make them ideally suitable both for cellular therapies and regenerative medicine. These include: relative ease of isolation, the ability to differentiate along mesenchymal and non-mesenchymal lineages in vitro and the ability to be extensively expanded in culture without a loss of differentiative capacity. MSC are not only hypoimmunogenic, but they mediate immunosuppression upon transplantation, and possess pronounced anti-inflammatory properties. They are able to home to damaged tissues, tumors, and metastases following systemic administration. The ability of homing holds big promise for tumor-targeted delivery of therapeutic agents. Viruses are naturally evolved vehicles efficiently transferring their genes into host cells. This ability made them suitable for engineering vector systems for the delivery of genes of interest. MSC can be retrovirally transduced with genes encoding prodrug-converting genes (suicide genes), which are not toxic per se, but catalyze the formation of highly toxic metabolites following the application of a nontoxic prodrug. The homing ability of MSC holds advantages compared to virus vehicles which display many shortcomings in effective delivery of the therapeutic agents. Gene therapies mediated by viruses are limited by their restricted ability to track cancer cells infiltrating into the surrounding tissue, and by their low migratory capacity towards tumor. Thus combination of cellular therapy and gene delivery is an attractive option - it protects the vector from immune surveillance, and supports targeted delivery of a therapeutic gene/protein to the tumor site.

  9. 3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line.

    Science.gov (United States)

    Hatta, Mitsutoki; Naganuma, Kaori; Kato, Kenichi; Yamazaki, Jun

    In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial-mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell-cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. [Effects of Sam68 gene silence on proliferation of acute T lymphoblastic leukemia cell line Jurkat].

    Science.gov (United States)

    Wang, Chi-Juan; Xu, Hua; Zhang, Hai-Rui; Wang, Jian; Lin, Ya-Ni; Pang, Tian-Xiang; Li, Qing-Hua

    2014-08-01

    This study was purpose to investigate the effect of Sam68 gene silence on proliferation of human acute T lymphoblastic leukemia cell line Jurkat. The sequence of shRNA targeting the site 531-552 of Sam68 mRNA was designed and chemically synthesized, then a single-vector lentiviral, Tet-inducible shRNA-Sam68 system (pLKO-Tet-On) was constructed; next the Jurkat cells were infected with lentivirus to create stable cell clones with regulatable Sam68 gene expression. The inhibitory efficiency of Sam68 gene was assayed by Real-time PCR and Western blot; the cell activity of Jurkat cells was detected with MTT assay; the change of colony forming potential of Jurkat cells was analyzed by colony forming test; the cell cycle distribution was tested by flow cytometry. The results indicated that the expression of Sam68 in experimental cells was statistically decreased as compared with that of the control cells; the cells activity and colony forming capacity of the Jurkat cells with Sam68 gene silence were significantly inhibited; with Sam68 gene silencing, the percentage of S phase cells was significantly increased, while the percentage of G2 phase cells was significantly decreased. It is concluded that the silencing Sam68 gene using shRNA interference can effectively inhibit the proliferation of human acute T lymphoblastic leukemia cell line Jurkat.

  11. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes.

    Science.gov (United States)

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

  12. PRDM1/BLIMP1: a tumor suppressor gene in B and T cell lymphomas.

    Science.gov (United States)

    Boi, Michela; Zucca, Emanuele; Inghirami, Giorgio; Bertoni, Francesco

    2015-05-01

    The gene encoding the human BLIMP1, prdm1, is located on chromosome 6q21, a locus frequently deleted in lymphoid tumors. BLIMP1 is able to silence its target genes in a context-dependent manner through different mechanisms. BLIMP1 is expressed in both B and T cells, in which it plays important functions. In B cells, BLIMP1 acts as the master regulator of plasma cell differentiation, repressed by BCL6 and repressing both BCL6 and PAX5. In T cells, BLIMP1 is a critical factor for most terminal effector cell differentiation in both CD4+ and CD8+ T cells. BLIMP1 is frequently inactivated in a variety of lymphomas, including diffuse large B cell lymphomas, Natural Killer cell lymphoma and anaplastic large T cell lymphoma. In this review, we will summarize the role of BLIMP1 in normal cells, focusing on lymphoid cells, and on its function as tumor suppressor gene in lymphomas.

  13. Identification of testosterone-/androgen receptor-regulated genes in mouse Sertoli cells

    Institute of Scientific and Technical Information of China (English)

    Qiao-Xia Zhang; Xiao-Yan Zhang; Zhen-Ming Zhang; Wei Lu; Ling Liu; Gang Li; Zhi-Ming Cai; Yao-Ting Gui; Chawnshang Chang

    2012-01-01

    Androgen and androgen receptor (AR) play important roles in male spermatogenesis and fertility,yet detailed androgenlAR signals in Sertoli cells remain unclear.To identify AR target genes in Sertoli cells,we analyzed the gene expression profiles of testis between mice lacking AR in Sertoli cells (S-AR-/y) and their littermate wild-type (WT) mice.Digital gene expression analysis identified 2276 genes downregulated and 2865 genes upregulated in the S-AR-/y mice testis compared to WT ones.To further nail down the difference within Sertoli cells,we first constructed Sertoli cell line TM4 with stably transfected AR (named as TM4/AR) and found androgens failed to transactivate AR in Sertoli TM4 and TM4/AR cells.Interestingly,additional transient transfection of AR-cDNA resulted in significant androgen responsiveness with TM4/AR cells showing 10 times more androgen sensitivity than TM4 cells.In the condition where maximal androgen response was demonstrated,we then analyzed gene expression and found the expression levels of 2313 genes were changed more than twofold by transient transfection of AR-cDNA in the presence of testosterone.Among these genes,603 androgen-/ AR-regulated genes,including 164 upregulated and 439 downregulated,were found in both S-AR-/y mice testis and TM4/AR cells.Using informatics analysis,the gene ontology was applied to analyze these androgen-/AR-regulated genes to predict the potential roles of androgen/AR in the process of spermatogenesis.Together,using gene analysis in both S-AR-/y mice testis and TM4/AR cells may help us to better understand the androgen/AR signals in Sertoli cells and their influences in spermatogenesis.

  14. Association Mapping of Cell Wall Synthesis Regulatory Genes and Cell Wall Quality in Switchgrass

    Energy Technology Data Exchange (ETDEWEB)

    Bartley, Laura [Univ. of Oklahoma, Norman, OK (United States). Dept. of Microbiology and Plant Biology; Wu, Y. [Oklahoma State Univ., Stillwater, OK (United States); Zhu, L. [Oklahoma State Univ., Stillwater, OK (United States); Brummer, E. C. [Noble Foundation, Ardmore, OK (United States); Saha, M. [Noble Foundation, Ardmore, OK (United States)

    2016-05-31

    Inefficient conversion of biomass to biofuels is one of the main barriers for biofuel production from such materials. Approximately half of polysaccharides in biomass remain unused by typical biochemical conversion methods. Conversion efficiency is influenced by the composition and structure of cell walls of biomass. Grasses such as wheat, maize, and rice, as well as dedicated perennial bioenergy crops, like switchgrass, make up ~55% of biomass that can be produced in the United States. Grass cell walls have a different composition and patterning compared with dicotyledonous plants, including the well-studied model plant, Arabidopsis. This project identified genetic determinants of cell wall composition in grasses using both naturally occurring genetic variation of switchgrass and gene network reconstruction and functional assays in rice. In addition, the project linked functional data in rice and other species to switchgrass improvement efforts through curation of the most abundant class of regulators in the switchgrass genome. Characterizing natural diversity of switchgrass for variation in cell wall composition and properties, also known as quality, provides an unbiased avenue for identifying biologically viable diversity in switchgrass cell walls. To characterizing natural diversity, this project generated cell wall composition and enzymatic deconstruction data for ~450 genotypes of the Switchgrass Southern Association Collection (SSAC), a diverse collection composed of 36 switchgrass accessions from the southern U.S. distribution of switchgrass. Comparing these data with other measures of cell wall quality for the same samples demonstrated the complementary nature of the diverse characterization platforms now being used for biomass characterization. Association of the composition data with ~3.2K single nucleotide variant markers identified six significant single nucleotide variant markers co-associated with digestibility and another compositional trait. These

  15. A rapid and efficient method to express target genes in mammalian cells by baculovirus

    Institute of Scientific and Technical Information of China (English)

    Tong Cheng; Chen-Yu Xu; Ying-Bin Wang; Min Chen; Ting Wu; Jun Zhang; Ning-Shao Xia

    2004-01-01

    AIM: To investigate the modification of baculovirus vector and the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of Spodoptera frugiperta (Sf9) cells infected by recombinant baculoviruses.METHODS: Two recombinant baculoviruses (BacV-CMVEGFPA, BacV-CMV-EGFPB) containing CMV-EGFP expression cassette were constructed. HepG2 cells were directly incubated with the culture supernatant of Sf9 cells infected by recombinant baculoviruses, and reporter gene transfer and expression efficiencies were analyzed by flow cytometry (FCM). The optimal transduction conditions were investigated by FCM assay in HepG2 cells. Gene-transfer and expression efficiencies in HepG2 or CV1 cells by baculovirus vectors were compared with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Twenty different mammalian cell lines were used to investigate the feasibility of delivering exogenous genes into different mammalian cells with the culture supernatant of infected Sf9 cells.RESULTS: CMV promoter could directly express reporter genes in Sf9 cells with a relatively low efficiency. Target cells incubated with the 1:1 diluted culture supernatant (moi=50) for 12 h at 37 ℃ could achieve the highest transduction and expression efficiencies with least impairment to cell viability. Under similar conditions the baculovirus vector could achieve the highest gene-transfer and expression efficiency than lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems. Most mammalian cell lines could be transduced with recombinant baculovirus. In primate adherent culture cells the recombinant baculovirus could arrive the highest infection and expression efficiencies, but it was not very satisfactory in the cell lines from mice and suspended culture cells.CONCLUSION: Mammalian cells incubated with the culture supernatant of infected Sf9 cells could serve as a very convenient way for rapid and efficient expression of foreign

  16. Global indiscriminate methylation in cell-specific gene promoters following reprogramming into human induced pluripotent stem cells.

    Science.gov (United States)

    Nissenbaum, Jonathan; Bar-Nur, Ori; Ben-David, Eyal; Benvenisty, Nissim

    2013-01-01

    Molecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm, and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global nonspecific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells.

  17. Gene expression profiles of the NCI-60 human tumor cell lines define molecular interaction networks governing cell migration processes.

    Directory of Open Access Journals (Sweden)

    Kurt W Kohn

    Full Text Available Although there is extensive information on gene expression and molecular interactions in various cell types, integrating those data in a functionally coherent manner remains challenging. This study explores the premise that genes whose expression at the mRNA level is correlated over diverse cell lines are likely to function together in a network of molecular interactions. We previously derived expression-correlated gene clusters from the database of the NCI-60 human tumor cell lines and associated each cluster with function categories of the Gene Ontology (GO database. From a cluster rich in genes associated with GO categories related to cell migration, we extracted 15 genes that were highly cross-correlated; prominent among them were RRAS, AXL, ADAM9, FN14, and integrin-beta1. We then used those 15 genes as bait to identify other correlated genes in the NCI-60 database. A survey of current literature disclosed, not only that many of the expression-correlated genes engaged in molecular interactions related to migration, invasion, and metastasis, but that highly cross-correlated subsets of those genes engaged in specific cell migration processes. We assembled this information in molecular interaction maps (MIMs that depict networks governing 3 cell migration processes: degradation of extracellular matrix, production of transient focal complexes at the leading edge of the cell, and retraction of the rear part of the cell. Also depicted are interactions controlling the release and effects of calcium ions, which may regulate migration in a spaciotemporal manner in the cell. The MIMs and associated text comprise a detailed and integrated summary of what is currently known or surmised about the role of the expression cross-correlated genes in molecular networks governing those processes.

  18. Expression profile of germ stem cell-specific genes in human spermatogonial stem cells after co culture with sertoli cells

    Directory of Open Access Journals (Sweden)

    Maria Zahiri

    2014-05-01

    Full Text Available Background: Human spermatogonial stem cells (SSCs, are the foundation of spermatogenesis. Because of low number and lack of significant marker in human SSCs, studying their characteristics, could provide better understanding about the biology of male fertility. This study was designed to examine the effects of in vitro co-culture with sertoli cells on SSC colonization and germ cells specific gene expression of human spermatogonial stem cells. Material and Methods: Testicular cells were isolated from testis biopsies by using two step enzymatic digestion and differential plating. two culture system were designed: co-culture with patient Sertoli cells and culture of SSC without co-culture(as control group. The number and diameter of colonies were evaluated during 3 weeks of culture. The expression of alpha 6 integrin, beta1 integrin and PLZF, as germ stem cell specific markers, was assessed using quantitative RT-PCR. Statistical analysis was performed using one way ANOVA in SPSS vesion 16 software with 95% Confidence interval . Result: Our results were showed that the number and diameter of colonies increased significantly in co-culture with sertoli cells (P<0.05. The expression profile of genes in 2nd and 3rd weeks of culture revealed that there is significant higher expression of germ stem cell markers in our co-culture group versus control group. Conclusion: Based on the optimal effects of sertoli cells on spermatogonial stem cells, co culture of the human SSCs with the feeder layer sertoli may be used as a suitable method for the enrichment of human spermatogonial stem cells.

  19. Nitric Oxide Prevents Mouse Embryonic Stem Cell Differentiation Through Regulation of Gene Expression, Cell Signaling, and Control of Cell Proliferation.

    Science.gov (United States)

    Tapia-Limonchi, Rafael; Cahuana, Gladys M; Caballano-Infantes, Estefania; Salguero-Aranda, Carmen; Beltran-Povea, Amparo; Hitos, Ana B; Hmadcha, Abdelkrim; Martin, Franz; Soria, Bernat; Bedoya, Francisco J; Tejedo, Juan R

    2016-09-01

    Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 μM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-β/β-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. J. Cell. Biochem. 117: 2078-2088, 2016. © 2016 Wiley Periodicals, Inc.

  20. Demethylation effects of elemene on the GSTP1 gene in HCC cell line QGY7703

    Science.gov (United States)

    WU, BAOQIANG; JIANG, YONG; ZHU, FENG; SUN, DONGLIN; HUANG, HONGJUN

    2016-01-01

    The present study aimed to investigate elemene's effects on cell proliferation, apoptosis, and the cell cycle in the hepatocellular carcinoma (HCC) cell line, QYG7703, and to investigate GSTP1 gene methylation change in QGY7703 cells after being treated with elemene to explore whether elemene reversed the abnormal GSTP1 gene methylation. QGY7703 cells were treated with different elemene concentrations. Cell proliferation was measured with MTT assay, cell apoptosis and cell cycle were analyzed by flow cytometry, and GSTP1 gene methylation was analyzed by methlation-specific polymerase chain reaction. The cells' apoptotic rate increased dose-dependently with elemene concentration, and the difference was statistically significant (P<0.05). Elemene treatment arrested the cells in S phase, and thus the percentage of cells in G1 phase decreased while the cells in S phase increased dose-dependently, and the difference was statistically significant compared to the control group (P<0.05). All QGY7703 cells were identified to contain GSTP1 gene methylation before being treated with elemene and the methylation state decreased after treatment. In the present study, elemene induced cell apoptosis, inhibited the cell cycle, and reversed GSTP1 gene methylation in QGY7703 cells. PMID:27073515

  1. Transcriptional expression of genes involved in cell invasion and migration by normal and tumoral trophoblast cells.

    Science.gov (United States)

    Janneau, Jean-Louis; Maldonado-Estrada, Juan; Tachdjian, Gérard; Miran, Isabelle; Motté, Nelly; Saulnier, Patrick; Sabourin, Jean-Christophe; Coté, Jean-François; Simon, Bénédicte; Frydman, René; Chaouat, Gérard; Bellet, Dominique

    2002-11-01

    Once initiated, invasion of trophoblast cells must be tightly regulated, particularly in early pregnancy. The mechanisms necessary for the invasion and migration of trophoblast cells are thought to be related to those involved in the invasive and metastatic properties of cancer cells. Quantitative PCR was used to measure, in trophoblast cells, the transcriptional expression profiles of four genes, INSL4, BRMS1, KiSS-1 and KiSS-1R, reported to be implicated in tumor invasion and metastasis. Laser capture microdissection and purification of trophoblast cells demonstrate that, as already known for INSL4, BRMS1, KiSS-1 and KiSS-1R are expressed by the trophoblast subset of placental tissues. Expression profiles of these genes studied in early placentas (7-9 weeks, n=55) and term placentas (n=11) showed that expression levels of BRMS1 are higher in term than in early placentas, while expression levels of KiSS-1R are higher in early than in term placentas. Low levels of expression of BRMS1 were observed in normal pregnancies, in molar pregnancies and in choriocarcinoma cell lines BeWo, JAR and JEG3 while, in striking contrast, the expression levels of INSL4, KiSS-1 and Kiss-1R were increased in both early placentas and molar pregnancies and were reduced in choriocarcinoma cells. These transcriptional expression profiles are in favor of a predominant role of INSL4, KiSS-1 and KiSS-1R in the control of the invasive and migratory properties of trophoblast cells.

  2. Identification of differentially expressed radiation-induced genes in cervix carcinoma cells using suppression subtractive hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun Sang; Lee, Young Sook; Lee, Jeung Hoon; Lee, Woong Hee; Seo, Eun Young; Cho, Moon June [Chungnam National University, Daejeon (Korea, Republic of)

    2005-03-15

    A number of genes and their products are induced early or late following exposure of cells to ionizing radiation. These radiation-induced genes have various effects of irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to identify the differentially expressed genes by radiation in cervix carcinoma cells. Total RNA and poly (A){sup +} mRNA were isolated from irradiated and non-irradiated HeLa cells. Forward-and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Of the 176 clones, 10 genes in the forward-subtracted library and 9 genes in the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. We identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

  3. Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells

    NARCIS (Netherlands)

    Chan, M.F.; van Amerongen, R.; Nijjar, T.; Cuppen, E.; Jones, P.A.; Laird, P.W.

    2001-01-01

    Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by c

  4. Altered gene expression profiles of NIH3T3 cells regulated by human lung cancer associated gene CT120

    Institute of Scientific and Technical Information of China (English)

    Xiang Huo HE; Jin Jun LI; Yi Hu XIE; Yun Tian TANG; Gen Fu YAO; Wen Xin QIN; Da Fang WAN; Jian Ren GU

    2004-01-01

    CT120, a novel membrane-associated gene implicated in lung carcinogenesis, was previously identified from chromosome 17p13.3 locus, a hot mutation spot involved in human malignancies. In the present study, we further determined that CT120 ectopic expression could promote cell proliferation activity of NIH3T3 cells using MTS assay, and monitored the downstream effects of CT120 in NIH3T3 cells with Atlas mouse cDNA expression arrays. Among 588known genes, 133 genes were found to be upregulated or downregulated by CT120. Two major signaling pathways involved in cell proliferation, cell survival and anti-apoptosis were overexpressed and activated in response to CT120:One is the Raf/MEK/Erk signal cascades and the other is the PI3K/Akt signal cascades, suggesting that CT120 might contribute, at least in part, to the constitutively activation of Erk and Akt in human lung caner cells. In addition, some tumor metastasis associated genes cathepsin B, cathepsin D, cathepsin L, MMP-2/TIMP-2 were also upregulated by CT120, upon which CT120 might be involved in tumor invasiveness and metastasis. In addition, CT120 might play an important role in tumor progression through modulating the expression of some candidate "Lung Tumor Progression"genes including B-Raf, Rab-2, BAX, BAG-1, YB-1, and Cdc42.

  5. Embryonic stem cell-derived microvesicles induce gene expression changes in Müller cells of the retina.

    Science.gov (United States)

    Katsman, Diana; Stackpole, Emma J; Domin, Daniel R; Farber, Debora B

    2012-01-01

    Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.

  6. Gene Delivery into Plant Cells for Recombinant Protein Production

    Directory of Open Access Journals (Sweden)

    Qiang Chen

    2015-01-01

    Full Text Available Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

  7. Precise gene modification mediated by TALEN and single-stranded oligodeoxynucleotides in human cells.

    Directory of Open Access Journals (Sweden)

    Xiaoling Wang

    Full Text Available The development of human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs facilitates in vitro studies of human disease mechanisms, speeds up the process of drug screening, and raises the feasibility of using cell replacement therapy in clinics. However, the study of genotype-phenotype relationships in ESCs or iPSCs is hampered by the low efficiency of site-specific gene editing. Transcription activator-like effector nucleases (TALENs spurred interest due to the ease of assembly, high efficiency and faithful gene targeting. In this study, we optimized the TALEN design to maximize its genomic cutting efficiency. We showed that using optimized TALENs in conjunction with single-strand oligodeoxynucleotide (ssODN allowed efficient gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb can be accomplished at high efficiencies. We established human tumor cell lines and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21 gene and miR-9-2 gene. These cell lines provide a robust platform to dissect the roles these genes play during cell differentiation and tumorigenesis. We also observed that the endogenous homologous chromosome can serve as a donor template for gene editing. Overall, our studies demonstrate the versatility of using ssODN and TALEN to establish genetically modified cells for research and therapeutic application.

  8. Space experiment "Rad Gene"-report 1; p53-Dependent gene expression in human cultured cells exposed to space environment

    Science.gov (United States)

    Takahashi, Akihisa; Ohnishi, Takeo; Suzuki, Hiromi; Omori, Katsunori; Seki, Masaya; Hashizume, Toko; Shimazu, Toru; Ishioka, Noriaki

    The space environment contains two major biologically significant influences: space radiations and microgravity. A p53 tumor suppressor protein plays a role as a guardian of the genome through the activity of p53-centered signal transduction pathways. The aim of this study was to clarify the biological effects of space radiations, microgravity and a space environment on the gene and protein expression of p53-dependent regulated genes. Space experiments were performed with two human cultured lymphoblastoid cell lines: one cells line (TSCE5) bears a wild-type p53 gene status, and another cells line (WTK1) bears a mutated p53 gene status. Un-der one gravity or microgravity condition, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station (ISS) for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples also were cultured for 8 days in the CBEF on the ground during the same periods as space flight. Gene and protein expression was analyzed by using DNA chip (a 44k whole human genome microarray, Agilent Technologies Inc.) and protein chip (PanoramaTM Ab MicroArray, Sigma-Aldrich Co.), respectively. In addition, we analyzed the gene expression in cultured cells after space flight during 133 days with frozen condition. We report the results and discussion from the viewpoint of the functions of the up-regulated and down-regulated genes after an exposure to space radiations and/or microgravity. The initial goal of this space experiment was completely achieved. It is expected that data from this type of work will be helpful in designing physical protection from the deleterious effects of space radiations during long term stays in space.

  9. The effects of antisense PTEN gene transfection on the growth and invasion of glioma cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-jie; ZHENG Zhao-cong; WANG Ru-mi; WANG Shou-sen; YANG Wei-zhong

    2006-01-01

    Objective:To study the effects of antisense PTEN gene on the growth and invasion of glioma cells. Methods:A pcDNA3. 1/Hygro (-) recombinant plasmid containing antisense PTEN gene fragment was constructed. Glioma cells of primary culture were transfected with antisense PTEN gene vector and stably transfected clones were selected. Then, the different growth and invasion abilities and the different MMP9 mRNA expressions of three kinds of cells were observed, including the transfected cells, untransfected cells and the cells transfected with empty vector. Results :The abilities of growth and invasion of the transfected cells and the expressions of MMP9 mRNA were obviously enhanced. Conclusion: Antisense PTEN gene could have a negative impact on the growth and invasion of primary culture glioma cells.

  10. DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN METABOLISM BETWEEN TUMORIGENITIC HUMAN LEUKEMIA CELL LINES K562 AND K562-n

    Institute of Scientific and Technical Information of China (English)

    吕书晴; 许小平; 夏放; 居小萍; 李瑶; 应康; 毛裕民

    2003-01-01

    Objective: To study the molecular mechanism of different tumorigenicity in nude mice of human leukemia cell lines K562-n and K562. Methods: To analyze the genes differently expressed between K562 and K562-n cells by using cDNA microarray technique. Results: Among the 12800 genes detected, some genes involved in material metabolism and material transport were differently expressed between K562-n and K562 cells. These genes include homo sapiens placenta-specific ATP-binding cassette transporter gene, dihydrodiol dehydrogenase gene, hepatic dihydrodiol dehydrogenase gene, NAD-dependent methylene tetrahydrofolate dehydrogenase cyclohydrolase, lysophosphatidic acid acyltransferase, alpha gene, argininosuccinate lyase gene, mitochondrial isocitrtate dehydrogenase, adhesion protein SQM1 gene, dimethylarginine dimethylamino-hydrolase gene, M1 subunit of ribonucleotide reductase and farnesyl pyrophosphate synthetase gene. Conclusion: The high tumorigenicity of K562-n cells is related to the different expression of some genes concerned with cell metabolism and material transpoert.

  11. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik

    2013-01-01

    of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE...... for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize......Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin...

  12. Spontaneously immortalised bovine mammary epithelial cells exhibit a distinct gene expression pattern from the breast cancer cells

    Directory of Open Access Journals (Sweden)

    Li Qianqian

    2010-10-01

    Full Text Available Abstract Background Spontaneous immortalisation of cultured mammary epithelial cells (MECs is an extremely rare event, and the molecular mechanism behind spontaneous immortalisation of MECs is unclear. Here, we report the establishment of a spontaneously immortalised bovine mammary epithelial cell line (BME65Cs and the changes in gene expression associated with BME65Cs cells. Results BME65Cs cells maintain the general characteristics of normal mammary epithelial cells in morphology, karyotype and immunohistochemistry, and are accompanied by the activation of endogenous bTERT (bovine Telomerase Reverse Transcriptase and stabilisation of the telomere. Currently, BME65Cs cells have been passed for more than 220 generations, and these cells exhibit non-malignant transformation. The expression of multiple genes was investigated in BME65Cs cells, senescent BMECs (bovine MECs cells, early passage BMECs cells and MCF-7 cells (a human breast cancer cell line. In comparison with early passage BMECs cells, the expression of senescence-relevant apoptosis-related gene were significantly changed in BME65Cs cells. P16INK4a was downregulated, p53 was low expressed and Bax/Bcl-2 ratio was reversed. Moreover, a slight upregulation of the oncogene c-Myc, along with an undetectable level of breast tumor-related gene Bag-1 and TRPS-1, was observed in BME65Cs cells while these genes are all highly expressed in MCF-7. In addition, DNMT1 is upregulated in BME65Cs. These results suggest that the inhibition of both senescence and mitochondrial apoptosis signalling pathways contribute to the immortality of BME65Cs cells. The expression of p53 and p16INK4a in BME65Cs was altered in the pattern of down-regulation but not "loss", suggesting that this spontaneous immortalization is possibly initiated by other mechanism rather than gene mutation of p53 or p16INK4a. Conclusions Spontaneously immortalised BME65Cs cells maintain many characteristics of normal BMEC cells and

  13. On the Role of PDZ Domain-Encoding Genes in Drosophila Border Cell Migration

    OpenAIRE

    Aranjuez, George; Kudlaty, Elizabeth; Longworth, Michelle S; McDonald, Jocelyn A.

    2012-01-01

    Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in v...

  14. Noncoding RNA small nucleolar RNA host gene 1 promote cell proliferation in nonsmall cell lung cancer

    Directory of Open Access Journals (Sweden)

    J You

    2014-01-01

    Full Text Available Background: Nonsmall cell lung cancer (NSCLC is the major cause of cancer death worldwide. Increasing evidence shows that noncoding RNAs (ncRNAs are widely involved in the development and progression of NSCLC. ncRNA small nucleolar RNA host gene 1 (SNHG1 has not been studied in cancer, especially its role in lung cancer remains unknown. Our studies were designed to investigate the expression and biological significance of SNHG1 in lung cancer. SNHG1 may be a novel ncRNA in early diagnosis in lung cancer. Methods: Noncoding RNA SNHG1 expression in 7 lung cancer cell lines was measured by quantitative real-time polymerase chain reaction. RNA interference approaches were used to find the biological functions of SNHG1. The effect of SNHG1 on proliferation was evaluated by cell count and crystal violet stains. Results: Noncoding RNA SNHG1 expression was significantly upregulated in lung cancer cells when compared with normal bronchial epithelial cells. In addition, in vitro assays our results indicated that knockdown of SNHG1 inhibited cell proliferation. Conclusions: Our data indicated that ncRNA SNHG1 is significantly upregulated in NSCLC cell lines and may represent a new biomarker and a potential therapeutic target for NSCLC intervention.

  15. Genes involved in cell adhesion and signaling: a new repertoire of retinoic acid receptor target genes in mouse embryonic fibroblasts.

    Science.gov (United States)

    Al Tanoury, Ziad; Piskunov, Aleksandr; Andriamoratsiresy, Dina; Gaouar, Samia; Lutzing, Régis; Ye, Tao; Jost, Bernard; Keime, Céline; Rochette-Egly, Cécile

    2014-02-01

    Nuclear retinoic acid (RA) receptors (RARα, β and γ) are ligand-dependent transcription factors that regulate the expression of a battery of genes involved in cell differentiation and proliferation. They are also phosphoproteins and we previously showed the importance of their phosphorylation in their transcriptional activity. In the study reported here, we conducted a genome-wide analysis of the genes that are regulated by RARs in mouse embryonic fibroblasts (MEFs) by comparing wild-type MEFs to MEFs lacking the three RARs. We found that in the absence of RA, RARs control the expression of several gene transcripts associated with cell adhesion. Consequently the knockout MEFs are unable to adhere and to spread on substrates and they display a disrupted network of actin filaments, compared with the WT cells. In contrast, in the presence of the ligand, RARs control the expression of other genes involved in signaling and in RA metabolism. Taking advantage of rescue cell lines expressing the RARα or RARγ subtypes (either wild-type or mutated at the N-terminal phosphorylation sites) in the null background, we found that the expression of RA-target genes can be controlled either by a specific single RAR or by a combination of RAR isotypes, depending on the gene. We also selected genes that require the phosphorylation of the receptors for their regulation by RA. Our results increase the repertoire of genes that are regulated by RARs and highlight the complexity and diversity of the transcriptional programs regulated by RARs, depending on the gene.

  16. Reversing effect of exogenous WWOX gene expression on malignant phenotype of primary cultured lung carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    ZHOU Yu-long; LI Yue-chuan; SHOU Feng; LIU Chang-qi; PU Yong; TANG Hua

    2010-01-01

    Background Whether WW domain containing oxidoreductase (WWOX) gene is a tumor-suppressor is still controversial. Some researchers found that the transcription of the WWOX gene was lacking not only in tumor tissues but also in non-tumorous tissues and sometimes in normal tissues. Hence it is important to explore the role of the expression of the exogenous WWOX gene in the proliferation and apoptosis of primary cultured lung carcinoma cells. Methods Lipofection technique was used to determine primary cultured lung carcinoma cells containing the highly expressed exogenous WWOX gene and primary cultured cells with vectors as controls. An animal model of lung cancer was made by subcutaneous implantation of tumor cells into nude mice. RT-PCR, Western blotting, flow cytometry, and TUN EL were used to detect the transcription, expression of the exogenous gene and the effect of the expression of targeted genes on the proliferation and apoptosis of the primary cultured lung carcinoma cells. Results The growth, clone formation rate (CFR) ((5.33±1.53)%) of the primary lung cancer cells transfected with the WWOX gene, tumor size and weight were significantly lower than those of the non-transfected lung cancer cells (CFR: (14.33±1.53)%) and the primary lung cancer cells transfected with blank plasmids (CFR: (11.00±1.73)%, P<0.05). The apoptosis level of primary lung cancer cells transfected with the WWOX gene ((40.72±5.20)%) was significantly higher than that of the non-transfected lung cancer cells ((2.76±0.02)%) and the primary lung cancer cells transfected with blank plasmids ((2.72±0.15)%, P<0.05). Conclusion The expression of the exogenous WWOX gene can significantly inhibit the proliferation of lung cancer cells and induce their apoptosis, suggesting that the WWOX gene possesses tumor-suppressing effect.

  17. GENE EXPRESSION PROFILING OF HUMAN PROMYELOCYTIC LEUKEMIA HL-60 CELL TREATED BY AJOENE

    Institute of Scientific and Technical Information of China (English)

    方志俊; 黄文秀; 黄明辉; 梁润松; 崔景荣; 王夔; 杨梦苏

    2002-01-01

    Objective: Ajoene, a major compound extracted from crashed garlic, has been shown to have antitumor, antimycotic, antimicrobial, antimutagenic functions in vivo or in vitro and treated as a potential antitumor drug. However, the molecular mechanisms underlying the tumor cytotoxicity of ajoene and even garlic substances are poorly defined. In the present study, we aimed to generate gene expression profiles of HL-60 cell treated by ajoene. Methods: A cDNA microarray presenting 2400 of genes amplified from human leukocyte cDNA library was constructed and the gene expression profiles of HL-60 cell induced by ajoene were generated. Results: After data analysis, 28 differentially expressed genes were identified and sequenced. These genes include 21 known genes and 7 ESTs. Most of the known genes are related to cell apoptosis, such as secretory granule (PRG1), beta-2 microglobulin (B2M), 16S ribosomal RNA gene and ribosomal protein S12. Several genes are related to cell differentiation, including the genes similar to H3 histone and ribosomal protein L31. Northern blot analysis was used to verify and quantify the expression of selected genes. Conclusion: Ajoene can induce HL-60 cell apoptosis significantly and may play a role in differentiation. cDNA microarray technology can be a valuable tool to gain insight into molecular events of pharmacological mechanism of herbal medicine.

  18. Changes in winter depression phenotype correlate with white blood cell gene expression profiles: a combined metagene and gene ontology approach.

    Science.gov (United States)

    Bosker, Fokko J; Terpstra, Peter; Gladkevich, Anatoliy V; Janneke Dijck-Brouwer, D A; te Meerman, Gerard; Nolen, Willem A; Schoevers, Robert A; Meesters, Ybe

    2015-04-03

    In the present study we evaluate the feasibility of gene expression in white blood cells as a peripheral marker for winter depression. Sixteen patients with winter type seasonal affective disorder were included in the study. Blood was taken by venous puncture at three time points; in winter prior and following bright light therapy and in summer. RNA was isolated, converted into cRNA, amplified and hybridized on Illumina® gene expression arrays. The raw optical array data were quantile normalized and thereafter analyzed using a metagene approach, based on previously published Affymetrix gene array data. The raw data were also subjected to a secondary analysis focusing on circadian genes and genes involved in serotonergic neurotransmission. Differences between the conditions were analyzed, using analysis of variance on the principal components of the metagene score matrix. After correction for multiple testing no statistically significant differences were found. Another approach uses the correlation between metagene factor weights and the actual expression values, averaged over conditions. When comparing the correlations of winter vs. summer and bright light therapy vs. summer significant changes for several metagenes were found. Subsequent gene ontology analyses (DAVID and GeneTrail) of 5 major metagenes suggest an interaction between brain and white blood cells. The hypothesis driven analysis with a smaller group of genes failed to demonstrate any significant effects. The results from the combined metagene and gene ontology analyses support the idea of communication between brain and white blood cells. Future studies will need a much larger sample size to obtain information at the level of single genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra; Peck, Matthew L.; Vega-Sánchez, Miguel E.; Williams, Brian; Chiniquy, Dawn M.; Saha, Prasenjit; Pattathil, Sivakumar; Conlin, Brian; Zhu, Lan; Hahn, Michael G.; Willats, William G. T.; Scheller, Henrik V.; Ronald, Pamela C.; Bartley, Laura E.

    2016-08-01

    Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.

  20. Identifying arsenic trioxide (ATO) functions in leukemia cells by using time series gene expression profiles.

    Science.gov (United States)

    Yang, Hong; Lin, Shan; Cui, Jingru

    2014-02-10

    Arsenic trioxide (ATO) is presently the most active single agent in the treatment of acute promyelocytic leukemia (APL). In order to explore the molecular mechanism of ATO in leukemia cells with time series, we adopted bioinformatics strategy to analyze expression changing patterns and changes in transcription regulation modules of time series genes filtered from Gene Expression Omnibus database (GSE24946). We totally screened out 1847 time series genes for subsequent analysis. The KEGG (Kyoto encyclopedia of genes and genomes) pathways enrichment analysis of these genes showed that oxidative phosphorylation and ribosome were the top 2 significantly enriched pathways. STEM software was employed to compare changing patterns of gene expression with assigned 50 expression patterns. We screened out 7 significantly enriched patterns and 4 tendency charts of time series genes. The result of Gene Ontology showed that functions of times series genes mainly distributed in profiles 41, 40, 39 and 38. Seven genes with positive regulation of cell adhesion function were enriched in profile 40, and presented the same first increased model then decreased model as profile 40. The transcription module analysis showed that they mainly involved in oxidative phosphorylation pathway and ribosome pathway. Overall, our data summarized the gene expression changes in ATO treated K562-r cell lines with time and suggested that time series genes mainly regulated cell adhesive. Furthermore, our result may provide theoretical basis of molecular biology in treating acute promyelocytic leukemia. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).

    Science.gov (United States)

    Varshney, N; Mohanty, A K; Kumar, S; Kaushik, J K; Dang, A K; Mukesh, M; Mishra, B P; Kataria, R; Kimothi, S P; Mukhopadhyay, T K; Malakar, D; Prakash, B S; Grover, S; Batish, V K

    2012-06-01

    We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), β-actin (ACTB), β-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, β-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation.

  2. THE EXPRESSION OF CONNEXIN GENES IN NASOPHARYNGEAL CARCINOMA CELLS AND THE EFFECT OF RETINOIC ACID ON THE REGULATION OF THOSE GENES

    Institute of Scientific and Technical Information of China (English)

    JIANG Ning; BIN Liang-hua; TANG Xiang-na; ZHOU Ming; ZENG Zhao-yang; Li Gui-yuan

    1999-01-01

    Objective: To detect which members in the connexin gene family are expressed in nasopharyngeal carcinoma (NPC) cell line HNE1, and the mechanism by which those genes are specifically switched on and off during retinoic acid (RA) induction. Methods: Establishing the cell growth curves of NPC cells. Observing the effect of RA on connexin genes by Northern hybridization. Results: Two genes Cx46 and Cx37, belonging to the connexin gene family, were expressed in HNE, The down-regulation of Cx46 and Cx37, up-regulation of RARa and growth inhibition was observed in HNE1, after exposure to RA. The gene expression and cell growth in HNE1 cells was restored after removal of RA. Conclusion: Two members of the connexin gene family: Cx37 and Cx46 were expressed in HNE1 cells, RA can inhibit the expression of those two genes mediated by RARa, and the effects of RA on HNE1 are reversible.

  3. Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms

    DEFF Research Database (Denmark)

    Svingen, T; Jørgensen, Anne; Rajpert-De Meyts, E

    2014-01-01

    expressed across the samples analysed: a so-called normalizing or housekeeping gene. Although this is a valid strategy, the identification of stable normalizing genes has proved challenging and a gene showing stable expression across all cells or tissues is unlikely to exist. Therefore, it is necessary...... to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers...

  4. Analysis of cell-type-specific gene expression during mouse spermatogenesis

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Nielsen, John E; Hansen, Martin Asser

    2004-01-01

    In rodents, changes in gene expression during spermatogenesis can be monitored by sampling testis from each day during postnatal development. However, changes in gene expression at the tissue level can reflect changes in the concentration of an mRNA in a specific cell type, changes in volume...... was gradually extinguished in the later spermatid stages but was followed by another cluster of genes expressed in spermatids. Finally, a group of genes was downregulated during spermatogenesis and probably expressed in nongerm cells. We believe that expression of most genes can be described by a combination...

  5. Modulation of Gene Expression Networks underlying Realgar-Induced Differentiation of Acute Promyelocytic Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    王怀宇; 刘陕西

    2002-01-01

    Objective: To elucidate the molecular mechanism of the differentiation of acute promyelocytic leukemia (APL) cell line NB4 induced by realgar. Methods: The response of NB4 cell to realgar was explored with a cDNA microarray representing 1003 different human genes. Results: The analysis of gene expression profiles indicated that 8 genes were up-regulated and 33 genes were down-regulated 48 hrs after realgar treatment. Among the 8 up-regulated genes, 2 genes were involved in ubiquitin proteasome degradation pathway. Some genes related to RNA processing, protein synthesis and signal transduction were down-regulated. Conclusion: The ubiquitin-proteasome degradation pathway may play an important role in the degradation of PML/RAR α fusion protein and the differentiation of NB4 cells.

  6. Measuring the toxic effects of high gene dosage on yeast cells.

    Science.gov (United States)

    Daniel, J

    1996-12-13

    A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed.

  7. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Høgh, Mette; Tannetta, D; Sargent, I

    2006-01-01

    directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. Design Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... the umbilical cords. Methods Gene expression was screened by Affymetrix GeneChips and confirmed with real-time polymerase chain reaction or enzyme-linked immunosorbent assay. Main outcome measures Fold changes in gene expression levels between treated and control cultures were calculated from the microarray...

  8. Effect of syncytiotrophoblast microvillous membrane treatment on gene expression in human umbilical vein endothelial cells

    DEFF Research Database (Denmark)

    Høgh, Mette; Tannetta, D; Sargent, I;

    2006-01-01

    directly causes the endothelial cell dysfunction of pre-eclampsia. This study investigates the effect of STBM on endothelial cell gene expression. Design Human umbilical vein endothelial cells were cultured in the presence and absence of STBM. At specified time points, total RNA was purified from...... the umbilical cords. Methods Gene expression was screened by Affymetrix GeneChips and confirmed with real-time polymerase chain reaction or enzyme-linked immunosorbent assay. Main outcome measures Fold changes in gene expression levels between treated and control cultures were calculated from the microarray...

  9. Analysis of cell growth and gene expression of porcine adipose tissue-derived mesenchymal stem cells as nuclear donor cell.

    Science.gov (United States)

    Oh, Hyun Ju; Park, Jung Eun; Park, Eun Jung; Kim, Min Jung; Kim, Geon A; Rhee, Sang Ho; Lim, Sang Hyun; Kang, Sung Keun; Lee, Byeong Chun

    2014-12-01

    In several laboratory animals and humans, adipose tissue-derived mesenchymal stem cells (ASC) are of considerable interest because they are easy to harvest and can generate a huge proliferation of cells from a small quantity of fat. In this study, we investigated: (i) the expression patterns of reprogramming-related genes in porcine ASC; and (ii) whether ASC can be a suitable donor cell type for generating cloned pigs. For these experiments, ASC, adult skin fibroblasts (AF) and fetal fibroblasts (FF) were derived from a 4-year-old female miniature pig. The ASC expressed cell-surface markers characteristic of stem cells, and underwent in vitro differentiation when exposed to specific differentiation-inducing conditions. Expression of DNA methyltransferase (DNMT)1 in ASC was similar to that in AF, but the highest expression of the DNMT3B gene was observed in ASC. The expression of OCT4 was significantly higher in FF and ASC than in AF (P development rate of cloned embryos derived from ASC was comparable to the development of those derived using FF. Total cell numbers of blastocysts derived using ASC and FF were significantly higher than in embryos made with AF. The results demonstrated that ASC used for SCNT have a potential comparable to those of AF and FF in terms of embryo in vitro development and blastocyst formation.

  10. DMPD: Activation of lymphokine genes in T cells: role of cis-acting DNA elements thatrespond to T cell activation signals. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 1492121 Activation of lymphokine genes in T cells: role of cis-acting DNA elements ...html) (.csml) Show Activation of lymphokine genes in T cells: role of cis-acting ...DNA elements thatrespond to T cell activation signals. PubmedID 1492121 Title Activation of lymphokine genes in T cells: role

  11. BMP2 gene delivery to bone mesenchymal stem cell by chitosan-g-PEI nonviral vector

    Science.gov (United States)

    Yue, Jianhui; Wu, Jun; Liu, Di; Zhao, Xiaoli; Lu, William W.

    2015-04-01

    Nanotechnology has made a significant impact on the development of nanomedicine. Nonviral vectors have been attracting more attention for the advantage of biosafety in gene delivery. Polyethylenimine (PEI)-conjugated chitosan (chitosan-g-PEI) emerged as a promising nonviral vector and has been demonstrated in many tumor cells. However, there is a lack of study focused on the behavior of this vector in stem cells which hold great potential in regenerative medicine. Therefore, in this study, in vitro gene delivering effect of chitosan-g-PEI was investigated in bone marrow stem cells. pIRES2-ZsGreen1-hBMP2 dual expression plasmid containing both the ZsGreen1 GFP reporter gene and the BMP2 functional gene was constructed for monitoring the transgene expression level. Chitosan-g-PEI-mediated gene transfer showed 17.2% of transfection efficiency and more than 80% of cell viability in stem cells. These values were higher than that of PEI. The expression of the delivered BMP2 gene in stem cells enhanced the osteogenic differentiation. These results demonstrated that chitosan-g-PEI is capable of applying in delivering gene to stem cells and providing potential applications in stem cell-based gene therapy.

  12. [Protection of corneal endothelium from apoptosis by gene and cell therapy].

    Science.gov (United States)

    Fuchsluger, T A

    2016-06-01

    Protection of corneal endothelium from apoptosis using gene and cell therapy is in a translational phase. This approach offers advantages for eye banking and after transplantation. Safe vehicles for gene or cell therapeutic transduction of corneal endothelium with nucleic acids are available. This strategy will be further developed in consultation with the Paul Ehrlich Institute and European regulatory authorities.

  13. Analysis of the DNDI gene in men with sporadic and familial testicular germ cell tumors

    NARCIS (Netherlands)

    Linger, Rachel; Dudakia, Darshna; Huddart, Robert; Tucker, Kathy; Friedlander, Michael; Phillips, Kelly-Anne; Hogg, David; Jewett, Michael A. S.; Lohynska, Radka; Daugaard, Gedske; Richard, Stephane; Chompret, Agnes; Stoppa-Lyonnet, Dominique; Bonaiti-Pellie, Catherine; Heidenreich, Axel; Albers, Peter; Olah, Edith; Geczi, Lajos; Bodrogi, Istvan; Daly, Peter A.; Guilford, Parry; Fossi, Sophie D.; Heimdal, Ketil; Tjulandin, Sergei A.; Liubchenko, Ludmila; Stoll, Hans; Weber, Walter; Einhorn, Lawrence; McMaster, Mary; Korde, Larissa; Greene, Mark H.; Nathanson, Katherine L.; Cortessis, Victoria; Easton, Douglas F.; Bishop, D. Timothy; Stratton, Michael R.; Rapley, Elizabeth A.

    2008-01-01

    A base substitution in the mouse DndI gene resulting in a truncated Dnd protein has been shown to be responsible for germ cell loss and the development of testicular germ cell tumors (TGCT) in the 129 strain of mice. We investigated the human orthologue of this gene in 263 patients (165 with a famil

  14. Systematic mapping of occluded genes by cell fusion reveals prevalence and stability of cis-mediated silencing in somatic cells

    Science.gov (United States)

    Looney, Timothy J.; Zhang, Li; Chen, Chih-Hsin; Lee, Jae Hyun; Chari, Sheila; Mao, Frank Fuxiang; Pelizzola, Mattia; Zhang, Lu; Lister, Ryan; Baker, Samuel W.; Fernandes, Croydon J.; Gaetz, Jedidiah; Foshay, Kara M.; Clift, Kayla L.; Zhang, Zhenyu; Li, Wei-Qiang; Vallender, Eric J.; Wagner, Ulrich; Qin, Jane Yuxia; Michelini, Katelyn J.; Bugarija, Branimir; Park, Donghyun; Aryee, Emmanuel; Stricker, Thomas; Zhou, Jie; White, Kevin P.; Ren, Bing; Schroth, Gary P.; Ecker, Joseph R.; Xiang, Andy Peng; Lahn, Bruce T.

    2014-01-01

    Both diffusible factors acting in trans and chromatin components acting in cis are implicated in gene regulation, but the extent to which either process causally determines a cell's transcriptional identity is unclear. We recently used cell fusion to define a class of silent genes termed “cis-silenced” (or “occluded”) genes, which remain silent even in the presence of trans-acting transcriptional activators. We further showed that occlusion of lineage-inappropriate genes plays a critical role in maintaining the transcriptional identities of somatic cells. Here, we present, for the first time, a comprehensive map of occluded genes in somatic cells. Specifically, we mapped occluded genes in mouse fibroblasts via fusion to a dozen different rat cell types followed by whole-transcriptome profiling. We found that occluded genes are highly prevalent and stable in somatic cells, representing a sizeable fraction of silent genes. Occluded genes are also highly enriched for important developmental regulators of alternative lineages, consistent with the role of occlusion in safeguarding cell identities. Alongside this map, we also present whole-genome maps of DNA methylation and eight other chromatin marks. These maps uncover a complex relationship between chromatin state and occlusion. Furthermore, we found that DNA methylation functions as the memory of occlusion in a subset of occluded genes, while histone deacetylation contributes to the implementation but not memory of occlusion. Our data suggest that the identities of individual cell types are defined largely by the occlusion status of their genomes. The comprehensive reference maps reported here provide the foundation for future studies aimed at understanding the role of occlusion in development and disease. PMID:24310002

  15. 40 CFR 798.5300 - Detection of gene mutations in somatic cells in culture.

    Science.gov (United States)

    2010-07-01

    ... cells in culture. 798.5300 Section 798.5300 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....5300 Detection of gene mutations in somatic cells in culture. (a) Purpose. Mammalian cell culture... selected by resistance to ouabain. (2) Description. Cells in suspension or monolayer culture are exposed to...

  16. CD133-targeted Gene Transfer Into Long-term Repopulating Hematopoietic Stem Cells

    NARCIS (Netherlands)

    Brendel, Christian; Goebel, Benjamin; Daniela, Abriss; Brugman, Martijn; Kneissl, Sabrina; Schwaeble, Joachim; Kaufmann, Kerstin B.; Mueller-Kuller, Uta; Kunkel, Hana; Chen-Wichmann, Linping; Abel, Tobias; Serve, Hubert; Bystrykh, Leonid; Buchholz, Christian J.; Grez, Manuel

    2015-01-01

    Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cell

  17. Reprogramming A375 cells to induced-resembled neuronal cells by structured overexpression of specific transcription genes

    OpenAIRE

    ZHANG, HENGZHU; Wei, Min; Jiang, Yangyang; Wang, Xiaodong; SHE, LEI; Yan, Zhengcun; Dong, Lun; Pang, Lujun; Wang, Xingdong

    2016-01-01

    Induced-resembled neuronal cells (irNCs) are generated by reprogramming human melanoma cells through the introduction of key transcription factors, providing novel concepts in the treatment of malignant tumor cells and making it possible to supply neural cells for laboratory use. In the present study, irNCs were derived from A375 cells by inducing the 'forced' overexpression of specific genes, including achaete-scute homolog 1 (Ascl1), neuronal differentiation factor 1 (Neurod1), myelin trans...

  18. Methylation patterns of immunoglobulin genes in lymphoid cells: correlation of expression and differentiation with undermethylation.

    Science.gov (United States)

    Storb, U; Arp, B

    1983-11-01

    Different states of eukaryotic gene expression are often correlated with different levels of methylation of DNA sequences containing structural genes and their flanking regions. To assess the potential role of DNA methylation in the expression of immunoglobulin genes, which require complex rearrangements prior to expression, methylation patterns were examined in cell lines representing different stages of lymphocyte maturation. Methylation of the second cytosine in the sequence 5' C-C-G-G 3' was determined by using Hpa II/Msp I endonuclease digestion. Four CH genes (C mu, C delta, C gamma 2b, and C alpha), C kappa, V kappa, C lambda, and V lambda genes were analyzed. The results lead to the following conclusions: (i) transcribed immunoglobulin genes are undermethylated; (ii) the C gene allelic to an expressed C gene is always also undermethylated; and (iii) all immunoglobulin loci tend to become increasingly undermethylated as B cells mature.

  19. Tumor-suppressor genes, cell cycle regulatory checkpoints, and the skin

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2015-01-01

    Full Text Available The cell cycle (or cell-division cycle is a series of events that take place in a cell, leading to its division and duplication. Cell division requires cell cycle checkpoints (CPs that are used by the cell to both monitor and regulate the progress of the cell cycle. Tumor-suppressor genes (TSGs or antioncogenes are genes that protect the cell from a single event or multiple events leading to cancer. When these genes mutate, the cell can progress to a cancerous state. We aimed to perform a narrative review, based on evaluation of the manuscripts published in MEDLINE-indexed journals using the Medical Subject Headings (MeSH terms "tumor suppressor′s genes," "skin," and "cell cycle regulatory checkpoints." We aimed to review the current concepts regarding TSGs, CPs, and their association with selected cutaneous diseases. It is important to take into account that in some cell cycle disorders, multiple genetic abnormalities may occur simultaneously. These abnormalities may include intrachromosomal insertions, unbalanced division products, recombinations, reciprocal deletions, and/or duplication of the inserted segments or genes; thus, these presentations usually involve several genes. Due to their complexity, these disorders require specialized expertise for proper diagnosis, counseling, personal and family support, and genetic studies. Alterations in the TSGs or CP regulators may occur in many benign skin proliferative disorders, neoplastic processes, and genodermatoses.

  20. Switch-like genes populate cell communication pathways and are enriched for extracellular proteins

    Directory of Open Access Journals (Sweden)

    Tozeren Aydin

    2008-01-01

    Full Text Available Abstract Background Recent studies have placed gene expression in the context of distribution profiles including housekeeping, graded, and bimodal (switch-like. Single-gene studies have shown bimodal expression results from healthy cell signaling and complex diseases such as cancer, however developing a comprehensive list of human bimodal genes has remained a major challenge due to inherent noise in human microarray data. This study presents a two-component mixture analysis of mouse gene expression data for genes on the Affymetrix MG-U74Av2 array for the detection and annotation of switch-like genes. Two-component normal mixtures were fit to the data to identify bimodal genes and their potential roles in cell signaling and disease progression. Results Seventeen percent of the genes on the MG-U74Av2 array (1519 out of 9091 were identified as bimodal or switch-like. KEGG pathways significantly enriched for bimodal genes included ECM-receptor interaction, cell communication, and focal adhesion. Similarly, the GO biological process "cell adhesion" and cellular component "extracellular matrix" were significantly enriched. Switch-like genes were found to be associated with such diseases as congestive heart failure, Alzheimer's disease, arteriosclerosis, breast neoplasms, hypertension, myocardial infarction, obesity, rheumatoid arthritis, and type I and type II diabetes. In diabetes alone, over two hundred bimodal genes were in a different mode of expression compared to normal tissue. Conclusion This research identified and annotated bimodal or switch-like genes in the mouse genome using a large collection of microarray data. Genes with bimodal expression were enriched within the cell membrane and extracellular environment. Hundreds of bimodal genes demonstrated alternate modes of expression in diabetic muscle, pancreas, liver, heart, and adipose tissue. Bimodal genes comprise a candidate set of biomarkers for a large number of disease states because

  1. Inhibition of cell proliferation, cell expansion and differentiation by the Arabidopsis SUPERMAN gene in transgenic tobacco plants.

    Science.gov (United States)

    Bereterbide, A; Hernould, M; Castera, S; Mouras, A

    2001-11-01

    Plant development depends upon the control of growth, organization and differentiation of cells derived from shoot and root meristems. Among the genes involved in flower organ determination, the cadastral gene SUPERMAN controls the boundary between whorls 3 and 4 and the growth of the adaxial outer ovule integument by down-regulating cell divisions. To determine the precise function of this gene we overexpressed ectopically the Arabidopsis thaliana (L.) Heynh. SUPERMAN gene in tobacco (Nicotiana tabacum L.). The transgenic plants exhibited a dwarf phenotype. Histologically and cytologically detailed analyses showed that dwarfism is correlated with a reduction in cell number, which is in agreement with the SUPERMAN function in Arabidopsis. Furthermore, a reduction in cell expansion and an impairment of cell differentiation were observed in tobacco organs. These traits were observed in differentiated vegetative and floral organs but not in meristem structures. A potential effect of the SUPERMAN transcription factor in the control of gibberellin biosynthesis is discussed.

  2. Global comparison of chromosome X genes of pulmonary telocytes with mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells, and lymphocytes.

    Science.gov (United States)

    Zhu, Yichun; Zheng, Minghuan; Song, Dongli; Ye, Ling; Wang, Xiangdong

    2015-09-28

    Telocytes (TCs) are suggested as a new type of interstitial cells with specific telopodes. Our previous study evidenced that TCs differed from fibroblasts and stem cells at the aspect of gene expression profiles. The present study aims to search the characters and patterns of chromosome X genes of TC-specific or TC-dominated gene profiles and fingerprints, investigate the network of principle genes, and explore potential functional association. We compared gene expression profiles in chromosome X of pulmonary TCs with mesenchymal stem cells (MSC), fibroblasts (Fb), alveolar type II cells (ATII), airway basal cells (ABC), proximal airway cells (PAC), CD8(+) T cells come from bronchial lymph nodes (T-BL), or CD8(+) T cells from lungs (T-L) by global analyses, and selected the genes which were consistently up or down regulated (>1 fold) in TCs compared to other cells as TC-specific genes. The functional and characteristic networks were identified and compared by bioinformatics tools. We selected 31 chromosome X genes as the TC-specific or dominated genes, among which 8 up-regulated (Flna, Msn, Cfp, Col4a5, Mum1l1, Rnf128, Syn1, and Srpx2) and 23 down-regulated (Abcb7, Atf1, Ddx26b, Drp2, Fam122b, Gyk, Irak1, Lamp2, Mecp2, Ndufb11, Ogt, Pdha1, Pola1, Rab9, Rbmx2, Rhox9, Thoc2, Vbp1, Dkc1, Nkrf, Piga, Tmlhe and Tsr2), as compared with other cells. Our data suggested that gene expressions of chromosome X in TCs are different with those in other cells in the lung tissue. According to the selected TC-specific genes, we infer that pulmonary TCs function as modulators which may enhance cellular growth and migration, resist senescence, protect cells from external stress, regulate immune responses, participate in tissue remodeling and repair, regulate neural function, and promote vessel formation.

  3. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Wei SHEN; Lingjiang MIN; Qingyu PAN; Yujiang SUN; Jixian DENG; Qingjie PAN

    2008-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.

  4. Differential Gene Expression in Thrombomodulin (TM; CD141)+ and TM− Dendritic Cell Subsets

    OpenAIRE

    Masaaki Toda; Zhifei Shao; Yamaguchi, Ken D.; Takehiro Takagi; Corina N D'Alessandro-Gabazza; Osamu Taguchi; Hugh Salamon; Leung, Lawrence L. K.; Gabazza, Esteban C.; John Morser

    2013-01-01

    Previously we have shown in a mouse model of bronchial asthma that thrombomodulin can convert immunogenic conventional dendritic cells into tolerogenic dendritic cells while inducing its own expression on their cell surface. Thrombomodulin(+) dendritic cells are tolerogenic while thrombomodulin(-) dendritic cells are pro-inflammatory and immunogenic. Here we hypothesized that thrombomodulin treatment of dendritic cells would modulate inflammatory gene expression. Murine bone marrow-derived de...

  5. Identification of G1-regulated genes in normally cycling human cells.

    Directory of Open Access Journals (Sweden)

    Maroun J Beyrouthy

    Full Text Available BACKGROUND: Obtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked. METHODOLOGY AND FINDINGS: We used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates (fos, atf3 and tceb by qPCR to further validate the newly identified genes. CONCLUSION AND SIGNIFICANCE: Genome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease.

  6. STUDY OF ECK GENE EXON-3 FROM HUMAN NORMAL TISSUE AND BREAST CANCER CELL LINE

    Institute of Scientific and Technical Information of China (English)

    李瑶琛; 孔令洪; 王一理; 司履生

    2003-01-01

    Objective To establish a method cloning the exon 3 of eck gene from normal tissue and ZR-75-1 cell line (a human breast cancer cell line)and study whether these genes exist mutant. Methods Designed a pair of specific primers and amplified the exon 3 of eck gene fragment from the extracted genomic DNA derived from normal epithelial cells from skin tissue and ZR-75-1 cell line respectively by PCR technique. Transformed the E.coil. JM109 with recombinant plamids constructed by inserting the amplified fragments into medium vector pUCm-T and sequenced these amplified fragments after primary screening of endonuclease restriction digestion and PCR amplification. Results ① Obtained the genomic DNA of human normal epithelial cells and ZR-75-1 cell line respectively. ② Obtained the amplified fragments of human exon 3 of eck gene through PCR technique. ③ Obtained the cloning vectors of exon 3 of eck gene of human normal epithelial cells and ZR-75-1 cell line respectively. ④ ZR-75-1 cell line exists mutation of nucleotides. Conclusion Successfully established the method of cloning the human exon 3 of eck gene and found some mutations in the detected samples. This study lays a foundation for further studying the function of eck gene in tumorgenesis.

  7. Gene Concepts in Higher Education Cell and Molecular Biology Textbooks

    Science.gov (United States)

    Albuquerque, Pitombo Maiana; de Almeida, Ana Maria Rocha; El-Hani, Nino Charbel

    2008-01-01

    Despite being a landmark of 20th century biology, the "classical molecular gene concept," according to which a gene is a stretch of DNA encoding a functional product, which may be a single polypeptide or RNA molecule, has been recently challenged by a series of findings (e.g., split genes, alternative splicing, overlapping and nested…

  8. EXPRESSION OF rhBMP-7 GENE IN TRANSDUCED BONE MARROW DERIVED STROMAL CELLS

    Institute of Scientific and Technical Information of China (English)

    段德宇; 杜靖远; 王洪; 刘勇; 郭晓东

    2002-01-01

    Objective. To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells(BMSCs). Methods. The marker gene , pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. Results. The exogenous gene could be expressed efficiently in transduced BMSCs. Conculsion. The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.

  9. Selenium is critical for cancer-signaling gene expression but not cell proliferation in human colon Caco-2 cells.

    Science.gov (United States)

    Zeng, Huawei; Botnen, James H

    2007-01-01

    Selenium (Se) is a potential anticarcinogenic nutrient, and the essential role of Se in cell growth is well recognized but certain cancer cells appear to have acquired a survival advantage under conditions of Se-deficiency. To understand the molecular basis of Se-anticancer effects at nutritional doses (nmol/L) for cultured cells, we generated Se-deficient colon Caco-2 cells by gradually reducing serum in media because serum contains a trace amount of Se. The glutathione peroxidase (GPx) activity of Se-deficient Caco-2 cells was 10.8 mU/mg protein compared to 133.6 approximately 146.3 mU/mg protein in Caco-2 cells supplemented with 500 nmol/L selenite, SeMSC or SeMet (three tested Se-chemical forms) after 7-d culture in serum free media. Interestingly, there were no detectable differences in cell growth, cell cycle progression between Se-deficient cells and cells supplemented with 500 nmol/L Se. To examine differential cancer signaling-gene expression between Se-deficient and Se-supplemented cells, we employed a cancer signal pathway-specific array assay coupled with the real time PCR analysis. Our data demonstrate that although Caco-2 cells are resistant to Se deprivation, Se may exert its anticancer property through increasing the expression of humoral defense gene (A2M) and tumor suppressor-related genes (IGFBP3, HHIP) while decreasing pro-inflammatory gene (CXC L9, HSPB2) expression.

  10. Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in CHO Cells

    Institute of Scientific and Technical Information of China (English)

    易继林; 田耕

    2003-01-01

    To clone the murine α-fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in CHO cells, total RNA were extracted from Hepa 1-6 cells, and then the murine α-fetoprotein gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. A fter transient transfection of CHO cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α-fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

  11. Gene expression profile changes in NB4 cells induced by realgar

    Institute of Scientific and Technical Information of China (English)

    王怀宇; 刘陕西; 吕晓虹; 赵晓艾; 陈思宇; 李信民

    2003-01-01

    Objectives To compare the gene expression profiles of acute promyelocytic leukemia cell line NB4 before and after 12 hours of realgar treatment using cDNA microarray.Methods Two cDNA probes were prepared through reverse transcription from mRNA of both untreated and realgar treated NB4 cells. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 1003 different human genes, and scanned for fluorescent intensity. The genes were screened through the analysis of the difference in two gene expression profiles. Results The analysis of gene expression profiles indicates that 9 genes were up-regulated and 37 genes were down-regulated. Among the 9 up-regulated genes, 2 genes were involved in a proteasome degradation pathway. Some genes related to protein synthesis, signal transduction and cell receptors were down-regulated. Conclusion PSMC2 and PSMD1 genes may play an important role in the apoptosis and partial differentiation of NB4 cells.

  12. Cell cycle networks link gene expression dysregulation, mutation, and brain maldevelopment in autistic toddlers.

    Science.gov (United States)

    Pramparo, Tiziano; Lombardo, Michael V; Campbell, Kathleen; Barnes, Cynthia Carter; Marinero, Steven; Solso, Stephanie; Young, Julia; Mayo, Maisi; Dale, Anders; Ahrens-Barbeau, Clelia; Murray, Sarah S; Lopez, Linda; Lewis, Nathan; Pierce, Karen; Courchesne, Eric

    2015-12-14

    Genetic mechanisms underlying abnormal early neural development in toddlers with Autism Spectrum Disorder (ASD) remain uncertain due to the impossibility of direct brain gene expression measurement during critical periods of early development. Recent findings from a multi-tissue study demonstrated high expression of many of the same gene networks between blood and brain tissues, in particular with cell cycle functions. We explored relationships between blood gene expression and total brain volume (TBV) in 142 ASD and control male toddlers. In control toddlers, TBV variation significantly correlated with cell cycle and protein folding gene networks, potentially impacting neuron number and synapse development. In ASD toddlers, their correlations with brain size were lost as a result of considerable changes in network organization, while cell adhesion gene networks significantly correlated with TBV variation. Cell cycle networks detected in blood are highly preserved in the human brain and are upregulated during prenatal states of development. Overall, alterations were more pronounced in bigger brains. We identified 23 candidate genes for brain maldevelopment linked to 32 genes frequently mutated in ASD. The integrated network includes genes that are dysregulated in leukocyte and/or postmortem brain tissue of ASD subjects and belong to signaling pathways regulating cell cycle G1/S and G2/M phase transition. Finally, analyses of the CHD8 subnetwork and altered transcript levels from an independent study of CHD8 suppression further confirmed the central role of genes regulating neurogenesis and cell adhesion processes in ASD brain maldevelopment.

  13. Gene expression profiling of CD8(+) T cells induced by ovarian cancer cells suggests a possible mechanism for CD8(+) Treg cell production.

    Science.gov (United States)

    Wu, Meng; Lou, Jianfang; Zhang, Shuping; Chen, Xian; Huang, Lei; Sun, Ruihong; Huang, Peijun; Pan, Shiyang; Wang, Fang

    2016-12-01

    The aim of this study was to investigate a possible mechanism of CD8(+) regulatory T-cell (Treg) production in an ovarian cancer (OC) microenvironment. Agilent microarray was used to detect changes in gene expression between CD8(+) T cells cultured with and without the SKOV3 ovarian adenocarcinoma cell line. QRT-PCR was performed to determine glycolysis gene expression in CD8(+) T cells from a transwell culturing system and OC patients. We also detected protein levels of glycolysis-related genes using Western blot analysis. Comparing gene expression profiles revealed significant differences in expression levels of 1420 genes, of which 246 were up-regulated and 1174 were down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis indicated that biological processes altered in CD8(+) Treg are particularly associated with energy metabolism. CD8(+) Treg cells induced by co-culture with SKOV3 had lower glycolysis gene expression compared to CD8(+) T cells cultured alone. Glycolysis gene expression was also decreased in the CD8(+) T cells of OC patients. These findings provide a comprehensive bioinformatics analysis of DEGs in CD8(+) T cells cultured with and without SKOV3 and suggests that metabolic processes may be a possible mechanism for CD8(+) Treg induction. © 2016 John Wiley & Sons Ltd.

  14. Application of HSVtk suicide gene to X-SCID gene therapy: ganciclovir treatment offsets gene corrected X-SCID B cells.

    Science.gov (United States)

    Uchiyama, Toru; Kumaki, Satoru; Ishikawa, Yoshinori; Onodera, Masafumi; Sato, Miki; Du, Wei; Sasahara, Yoji; Tanaka, Nobuyuki; Sugamura, Kazuo; Tsuchiya, Shigeru

    2006-03-10

    Recently, a serious adverse effect of uncontrolled clonal T cell proliferation due to insertional mutagenesis of retroviral vector was reported in X-SCID gene therapy clinical trial. To offset the side effect, we have incorporated a suicide gene into therapeutic retroviral vector for selective elimination of transduced cells. In this study, B-cell lines from two X-SCID patients were transduced with bicistronic retroviral vector carrying human gamma c chain cDNA and Herpes simplex virus thymidine kinase gene. After confirmation of functional reconstitution of the gamma c chain, the cells were treated with ganciclovir (GCV). The gamma c chain positive cells were eliminated under low concentration without cytotoxicity on untransduced cells and have not reappeared at least for 5 months. Furthermore, the gamma c chain transduced cells were still sensitive to GCV after five months. These results demonstrated the efficacy of the suicide gene therapy although further in vivo studies are required to assess feasibility of this approach in clinical trial.

  15. Differentiation of embryonic stem cells transfected by ibeB gene

    Institute of Scientific and Technical Information of China (English)

    SHANG Deshu; FANG Wengang; CHEN Yuhua

    2005-01-01

    We have previously identified an E. coli deter- minant, ibeB gene locus contributing to invasion of human brain microvascular endothelial cells. In the present study, we established embryonic stem (ES) cell lines overexpressing IbeB and found that exogenic ibeB gene could start-up expression of a neural stem cell specific marker, nestin, and give rise to polar changes. In analysis of IbeB location, it was found that GFP-IbeB fusion protein targeted at the ES cell nucleus. These data suggests that ibeB gene may play an important role in the regulation of nestin expression.

  16. Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, Toshiaki; Ikeda, Kazuhiro; Horie-Inoue, Kuniko [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Inoue, Satoshi, E-mail: INOUE-GER@h.u-tokyo.ac.jp [Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan); Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo 113-8655 (Japan)

    2014-09-26

    Highlights: • APP knockdown reduced proliferation and migration of prostate cancer cells. • APP knockdown reduced expression of metalloproteinase and EMT-related genes. • APP overexpression promoted LNCaP cell migration. • APP overexpression increased expression of metalloproteinase and EMT-related genes. - Abstract: Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APP has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.

  17. MICROARRAY ANALYSIS OF DIFFERENT GENE EXPRESSION OF HUMAN CERVICAL CANCER SUBCLONE CELL LINES

    Institute of Scientific and Technical Information of China (English)

    Chen Wei; Li Xu; Wang Xiang

    2006-01-01

    Objective To examine the differentially expressed invasion-related genes in two anchorage-independent uterine cervical carcinoma cell lines derived from the same patient using a cDNA array. Methods Two human uterine cervical carcinoma subclonal cell lines CS03 and CS07 derived from a single donor line CS1213 were established by limited dilution procedure. The two cDNA samples retro-transcribed from total RNA derived from CS03 and CS07 cells were screened by a cDNA microarray carrying 234 human cell-cycle related genes and 1011 human signal transduction and membrane receptor -associated genes, scanned with a ScanArray 3000 laser scanner. Results The cDNA microarray analysis showed that 12 genes in CS03 were up-regulated compared to CS07, and 24 genes in CS07 were up-regulated. The function of a number of differentially expressed genes was consistently associated with cell-cycle, cell proliferation, migration, apoptosis, signal transduction and tumor metastasis, including p34cdc2, TSC22, plasminogen activator inhibitor I (PAI-1)and desmosome associated protein(Pinin). Conclusion Multiple genes are differentially expressed in uterine cervical carcinoma cell lines even came from the same patient. It is suggested that these genes are involved in the different phenotypic characteristics and development of cervical carcinoma.

  18. THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Li Jian; Jiang Lei; Xia Yongjing; Li Hongxia; Hu Yajun; Hu Shixue; Xu Hongji

    1998-01-01

    Objective:To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene,on the growth of lung adenocarcinoma cell line GLC-82 and explore a gene therapy approach for lung adenocarcinoma. Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method. Infection effects were detected by biochemical staining of β-gal and immunohistochemical analysis of Rb protein. The Rb cDNA of infected cells were determined by PCR. The cell growth rate and cell cycle were observed by cell-counting and flow cytometry. Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein. The transfection of wild-type Rb gene could suppress GLC-82 cell proliferation and decrease the cellular DNA synthesis. Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer.

  19. MOLECULAR GENETIC DISORDERS IN THE VHL GENE AND METHYLATION OF SOME SUPPRESSOR GENES IN SPORADIC CLEAR-CELL RENAL CARCINOMAS

    Directory of Open Access Journals (Sweden)

    D. S. Mikhailenko

    2014-07-01

    Full Text Available Renal carcinoma (RC is one of ten most common malignancies in adults and an urgent problem of modern oncology. The purpose of the study was to make a molecular genetic analysis of a number of suppressor genes in RC, which was aimed at searching for and characterizing the potential markers of the disease. Two hundred and nine RC samples were examined, of them there were 192 clear-cell carcinomas. VHL gene mutations were detected by single-strand conformation polymorphism and sequence analyses while the methylation of suppressor genes was by the methylation-sensitive polymerase chain reaction. Somatic VHL mutations were determined in 35.4% of cases of clear-cell RC (CCRC. VHL gene disorders were found in 53.7% of patients with Stage 1, which counts in favor of VHL inactivation in early-stage CCRC. The methylation of the VHL, RASSF1, FHIT, and CDH1 genes was identified in 12, 56, 58.4, and 46.4% of primary tumors, respectively; that of at least one gene was in 84.1% of the samples. The hypermethylation of the RASSF1 gene was associated with late stages (p = 0.015 and the presence of metastases (p = 0.036; that of the CDH1 gene was related to the progression, invasion, and dissemination of primary tumors (p = 0.009, 0.039, and 0.002, respectively. The findings show it possible to use an analysis of abnormalities in the VHL gene and the methylation of the RASSF1 and CDH1 genes to develop a system of molecular genetic markers of RC.

  20. Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Zhaohua PEng [Mississippi State University; Ronald, Palmela [UC-Davis; Wang, Guo-Liang [The Ohio State University

    2013-04-26

    This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for each gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall

  1. Limited gene expression variation in human embryonic stem cell and induced pluripotent stem cell-derived endothelial cells.

    Science.gov (United States)

    White, Mark P; Rufaihah, Abdul J; Liu, Lei; Ghebremariam, Yohannes T; Ivey, Kathryn N; Cooke, John P; Srivastava, Deepak

    2013-01-01

    Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes, yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified, homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here, we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized, and defined growth factors were used to generate KDR(+) EC progenitors. Magnetic purification of a KDR(+) progenitor subpopulation resulted in an expanding, homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders.

  2. THE EFFECT OF TRANSFECTED CX43 GENE ON THE GJIC AND PROLIFERATION OF GLIOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    浦佩玉; 夏之柏; 黄强; 王春艳; 王广秀

    2002-01-01

    Objective: To evaluate the effect of Cx43 gene on gap junction intercellular communication (GJIC) and proliferation of glioma cells. Methods: Cx43 cDNA was transfected into TJ905 human glioblastoma cells using lipofectamine. The expression of Cx43 was identified by Northern blot analyses, in situ hybridization and immunohistochemistry. MTT assay and average number of AgNORs (Argyrophlic nuclear organizer regions) were used to determine the cell proliferation. TUNEL method was used for detection of cell apoptosis, and scrape loading and dye tranfer method for examination of GJIC. Results: The Cx43 expression was greatly upregulated when Cx43 gene was transfected into TJ905 glioma cells. The cell proliferation was inhibited while the cell apoptosis was not increased and GJIC was significantly restored in the glioma cells tranfected with Cx43 gene. Conclusion: Cx43 gene has an inhibitory effect on the glioma cell proliferation, but no effect on induction of cell apoptosis. The restoration of GJIC may be the major mechanism involved in its effect. Cx43 gene can be the candidate for gene therapy of gliomas.

  3. In tobacco BY-2 cells xyloglucan oligosaccharides alter the expression of genes involved in cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

    Science.gov (United States)

    González-Pérez, Lien; Perrotta, Lara; Acosta, Alexis; Orellana, Esteban; Spadafora, Natasha; Bruno, Leonardo; Bitonti, Beatrice M; Albani, Diego; Cabrera, Juan Carlos; Francis, Dennis; Rogers, Hilary J

    2014-10-01

    Xyloglucan oligosaccharides (XGOs) are breakdown products of XGs, the most abundant hemicelluloses of the primary cell walls of non-Poalean species. Treatment of cell cultures or whole plants with XGOs results in accelerated cell elongation and cell division, changes in primary root growth, and a stimulation of defence responses. They may therefore act as signalling molecules regulating plant growth and development. Previous work suggests an interaction with auxins and effects on cell wall loosening, however their mode of action is not fully understood. The effect of an XGO extract from tamarind (Tamarindus indica) on global gene expression was therefore investigated in tobacco BY-2 cells using microarrays. Over 500 genes were differentially regulated with similar numbers and functional classes of genes up- and down-regulated, indicating a complex interaction with the cellular machinery. Up-regulation of a putative XG endotransglycosylase/hydrolase-related (XTH) gene supports the mechanism of XGO action through cell wall loosening. Differential expression of defence-related genes supports a role for XGOs as elicitors. Changes in the expression of genes related to mitotic control and differentiation also support previous work showing that XGOs are mitotic inducers. XGOs also affected expression of several receptor-like kinase genes and transcription factors. Hence, XGOs have significant effects on expression of genes related to cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

  4. Acceleration of Apoptosis by Transfection of Bak Gene in Multi-drug Resistant Bladder Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    LIUYing; ZENGFuqing

    2004-01-01

    To study the killing effects of bak gene on multi-drug resistant (MDR) bladder cancer cells and the mechanisms. Methods: Bak gene was transfected into MDR bladder cancer cells by liposome. The expression of bak and Bcl-2 mRNA was detected by in situ hybridization. The expression of bak and Bcl-2 proteins was detected by SABC immunohistochemistry. The growth rate of human bladder cancer cells was studied by constructing the growth curve, cell apoptosis was measured by flow cytometry, and the morphology of cells was observed by fluorescence stain. Results: The expression of bak mRNA was positive in EJ/bak cells (P<0.05). Bak protein expression of EJ/bak cells was positive and Bcl-2 protein expression was decreased (P<0.05). The growth of MDR bladder cancer cells was significantly inhibited after bak gene was transfected (P<0.05). Apoptosis cells were increased significantly. The apoptosis rate was 35%. Apoptotic bodies can be found in these cells by fluorescence stain. Conclusion: Bak gene could inhibit the growth of MDR bladder cancer cells effectively. Inducing cell apoptosis by down-regulating the expression of Bcl-2 gene might be one of its mechanisms.

  5. Gene expression profiles of human liver cells mediated by hepatitis B virus X protein

    Institute of Scientific and Technical Information of China (English)

    Wei-ying ZHANG; Fu-qing XU; Chang-liang SHAN; Rong XIANG; Li-hong YE; Xiao-dong ZHANG

    2009-01-01

    Aim: To demonstrate the gene expression profiles mediated by hepatitis B virus X protein (HBx), we characterized the molecular features of pathogenesis associated with HBx in a human liver cell model.Methods: We examined gene expression profiles in L-O2-X cells, an engineered L-O2 cell line that constitutively expresses HBx, relative to L-O2 cells using an Agilent 22 K human 70-mer oligonucleotide microarray representing more than 21,329 unique, well-characterized Homo sapiens genes, Western blot analysis and RNA interference (RNAi) targeting HBx mRNA validated the overexpression of proliferating cell nuclear antigen (PCNA) and Bcl-2 in L-O2-X cells. Meanwhile, the BrdU incorporation assay was used to test cell proliferation mediated by upregulated cyclooxygenase-2 (COX-2).Results: The microarray showed that the expression levels of 152 genes were remarkably altered; 82 of the genes were upregulated and 70 genes were downregulated in L-O2-X cells. The altered genes were associated with signal transduction pathways, cell cycle, metastasis, transcriptional regulation, immune response, metabolism, and other processes. PCNA and Bcl-2 were upregulated in L-O2-X cells. Furthermore, we found that COX-2 upregulation in L-O2-X cells enhanced proliferation using the BrdU incorporation assay, whereas indomethacin (an inhibitor of COX-2) abolished the promotion.Conclusion: Our findings provide new evidence that HBx is able to regulate many genes that may be involved in the car-cinogenesis. These regulated genes mediated by HBx may serve as molecular targets for the prevention and treatment of hepatocellular carcinoma.

  6. Specific silencing of the REST target genes in insulin-secreting cells uncovers their participation in beta cell survival.

    Directory of Open Access Journals (Sweden)

    David Martin

    Full Text Available The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice, and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.

  7. Identification of stable reference genes in differentiating human pluripotent stem cells.

    Science.gov (United States)

    Holmgren, Gustav; Ghosheh, Nidal; Zeng, Xianmin; Bogestål, Yalda; Sartipy, Peter; Synnergren, Jane

    2015-06-01

    Reference genes, often referred to as housekeeping genes (HKGs), are frequently used to normalize gene expression data based on the assumption that they are expressed at a constant level in the cells. However, several studies have shown that there may be a large variability in the gene expression levels of HKGs in various cell types. In a previous study, employing human embryonic stem cells (hESCs) subjected to spontaneous differentiation, we observed that the expression of commonly used HKG varied to a degree that rendered them inappropriate to use as reference genes under those experimental settings. Here we present a substantially extended study of the HKG signature in human pluripotent stem cells (hPSC), including nine global gene expression datasets from both hESC and human induced pluripotent stem cells, obtained during directed differentiation toward endoderm-, mesoderm-, and ectoderm derivatives. Sets of stably expressed genes were compiled, and a handful of genes (e.g., EID2, ZNF324B, CAPN10, and RABEP2) were identified as generally applicable reference genes in hPSCs across all cell lines and experimental conditions. The stability in gene expression profiles was confirmed by reverse transcription quantitative PCR analysis. Taken together, the current results suggest that differentiating hPSCs have a distinct HKG signature, which in some aspects is different from somatic cell types, and underscore the necessity to validate the stability of reference genes under the actual experimental setup used. In addition, the novel putative HKGs identified in this study can preferentially be used for normalization of gene expression data obtained from differentiating hPSCs. Copyright © 2015 the American Physiological Society.

  8. Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

    DEFF Research Database (Denmark)

    Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P

    2010-01-01

    Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES...... cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression...... of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response...

  9. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Jared Carlson-Stevermer

    2016-01-01

    Full Text Available CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  10. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

    Science.gov (United States)

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-12

    CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  11. Age-related gene expression in luminal epithelial cells is driven by a microenvironment made from myoepithelial cells.

    Science.gov (United States)

    Miyano, Masaru; Sayaman, Rosalyn W; Stoiber, Marcus H; Lin, Chun-Han; Stampfer, Martha R; Brown, James B; LaBarge, Mark A

    2017-10-09

    Luminal epithelial cells in the breast gradually alter gene and protein expression with age, appearing to lose lineage-specificity by acquiring myoepithelial-like characteristics. We hypothesize that the luminal lineage is particularly sensitive to microenvironment changes, and age-related microenvironment changes cause altered luminal cell phenotypes. To evaluate the effects of different microenvironments on the fidelity of epigenetically regulated luminal and myoepithelial gene expression, we generated a set of lineage-specific probes for genes that are controlled through DNA methylation. Culturing primary luminal cells under conditions that favor myoepithelial propogation led to their reprogramming at the level of gene methylation, and to a more myoepithelial-like expression profile. Primary luminal cells' lineage-specific gene expression could be maintained when they were cultured as bilayers with primary myoepithelial cells. Isogenic stromal fibroblast co-cultures were unable to maintain the luminal phenotype. Mixed-age luminal-myoepithelial bilayers revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. We provide evidence that the luminal epithelial phenotype is exquisitely sensitive to microenvironment conditions, and that states of aging are cell non-autonomously communicated through microenvironment cues over at least one cell diameter.

  12. General approach for in vivo recovery of cell type-specific effector gene sets.

    Science.gov (United States)

    Barsi, Julius C; Tu, Qiang; Davidson, Eric H

    2014-05-01

    Differentially expressed, cell type-specific effector gene sets hold the key to multiple important problems in biology, from theoretical aspects of developmental gene regulatory networks (GRNs) to various practical applications. Although individual cell types of interest have been recovered by various methods and analyzed, systematic recovery of multiple cell type-specific gene sets from whole developing organisms has remained problematic. Here we describe a general methodology using the sea urchin embryo, a material of choice because of the large-scale GRNs already solved for this model system. This method utilizes the regulatory states expressed by given cells of the embryo to define cell type and includes a fluorescence activated cell sorting (FACS) procedure that results in no perturbation of transcript representation. We have extensively validated the method by spatial and qualitative analyses of the transcriptome expressed in isolated embryonic skeletogenic cells and as a consequence, generated a prototypical cell type-specific transcriptome database.

  13. Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells.

    Science.gov (United States)

    Patnala, Radhika; Lee, Sung-Hun; Dahlstrom, Jane E; Ohms, Stephen; Chen, Long; Dheen, S Thameem; Rangasamy, Danny

    2014-01-01

    Long Interspersed Elements (L1 elements) are biologically active retrotransposons that are capable of autonomous replication using their own reverse transcriptase (RT) enzyme. Expression of the normally repressed RT has been implicated in cancer cell growth. However, at present, little is known about the expression of L1-encoded RT activity or the molecular changes that are associated with RT activity in the development of breast cancer. Here, we report that RT activity is widespread in breast cancer cells. The expression of RT protein decreased markedly in breast cancer cells after treatment with the antiretroviral drug, efavirenz. While the majority of cells showed a significant reduction in proliferation, inhibition of RT was also accompanied by cell-specific differences in morphology. MCF7 cells displayed elongated microtubule extensions that adhered tightly to their substrate, while a large fraction of the T47D cells that we studied formed long filopodia projections. These morphological changes were reversible upon cessation of RT inhibition, confirming their dependence on RT activity. We also carried out gene expression profiling with microarrays and determined the genes that were differentially expressed during the process of cellular differentiation. Genes involved in proliferation, cell migration, and invasive activity were repressed in RT-inhibited cells. Concomitantly, genes involved in cell projection, formation of vacuolar membranes, and cell-to-cell junctions were significantly upregulated in RT-inhibited cells. qRT-PCR examination of the mRNA expression of these genes in additional cell lines yielded close correlation between their differential expression and the degree of cellular differentiation. Our study demonstrates that the inhibition of L1-encoded RT can reduce the rate of proliferation and promote differentiation of breast cancer cells. Together, these results provide a direct functional link between the expression of L1 retrotransposons and

  14. RETROVIRAL MEDIATED EFFICIENT TRANSFER ANDEXPRESSION OF MULTIPLE DRUG RESISTANCE GENE TO HUMAN LEUKEMIC CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%~ 72.5%) than in supernatant system (33.1%~ 46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

  15. Compact disk (CD)-shaped device for single cell isolation and PCR of a specific gene in the isolated cell.

    Science.gov (United States)

    Furutani, Shunsuke; Nagai, Hidenori; Takamura, Yuzuru; Kubo, Izumi

    2010-12-01

    For immediate discrimination among isolated cells we propose a novel device and technique for isolation of cells and sequential detection of specific gene(s) within them by polymerase chain reaction (PCR). In this study, we isolated Salmonella enterica cells and detected the Salmonella-specific invA gene from isolated cells by PCR on a compact disk (CD)-shaped device. This device enabled liquid flow by centrifugal force without a micro pump, and was fabricated from silicon wafer and glass to avoid evaporation of a small amount of reagent. One device has 24 microchannels, and 313 microchambers integrated on each microchannel. One microliter of PCR mixture containing cells was separated into microchambers on the device at 5000 rpm for 30 s. Each microchamber contained approximately 1.5 nL PCR mixture. A Poisson distribution of S. enterica cells was observed for different densities of cell suspension. At 200 cells μL(-1) of S. enterica or less, isolated single cells could be determined on the device by amplification of DNA of the invA gene; at 400 cells μL(-1), chambers containing no, one, two, or three cells could be determined on the device. Selective detection of S. enterica was achieved by PCR from a mixture of S. enterica and Escherichia coli on the CD-shaped device.

  16. Involvement of gene methylation changes in the differentiation of human amniotic epithelial cells into islet-like cell clusters.

    Science.gov (United States)

    Peng, Lin; Wang, Jian; Lu, Guangxiu

    2014-09-01

    Insulin-dependent diabetes results from destruction of the insulin-producing β-cells of the pancreas. Islet cell transplantation is a promising cure for diabetes. Here, we induced human amniotic epithelial cells (hAECs) to differentiate into islet-like cell clusters by nicotinamide plus betacellulin in vitro, and further investigated the DNA methylation status by a Nimble MeDIP microarray before and after cell differentiation to shed light on the molecular mechanisms of this differentiation. In addition, 5-Aza-2'-deoxycytidine was used to investigate whether the differentiation of hAECs into islet-like cells occurred through demethylation. Purified hAECs (CK18(+)/E-cadherin(+)/CD29(+)/CD90(-)/CD34(-)/CD45(-)) were isolated from human amnia. After induction, hAECs were found to be insulin positive and sensitive to glucose, indicating successful induction to islet-like cells. The methylation status of cell cytoskeleton-related genes was down-regulated and that of negative regulation of cell adhesion-related genes was up-regulated. The methylation status of pancreas development-related genes such as HNF1α and DGAT1 was decreased in hAECs after induction. After brief demethylation, INS gene expression was up-regulated in islet-like cell clusters, suggesting that DNA methylation changes were associated with the differentiation of hAECs into islet-like cell clusters.

  17. Parallel differentiation of embryonic stem cells into different cell types by a single gene-based differentiation system.

    Science.gov (United States)

    Thoma, Eva C; Maurus, Katja; Wagner, Toni U; Schartl, Manfred

    2012-04-01

    The generation of defined somatic cell types from pluripotent stem cells represents a promising system for many applications for regenerative therapy or developmental studies. Certain key developmental genes have been shown to be able to influence the fate determination of differentiating stem cells suggesting an alternative differentiation strategy to conventional medium-based methods. Here, we present a system allowing controlled, directed differentiation of embryonic stem cells (ESCs) solely by ectopic expression of single genes. We demonstrate that the myogenic master regulator myoD1 is sufficient to induce formation of skeletal muscle. In contrast to previous studies, our data suggest that myoD1-induced differentiation is independent of additional differentiation-inducing or lineage-promoting signals and occurs even under pluripotency-promoting conditions. Moreover, we demonstrate that single gene-induced differentiation enables the controlled formation of two distinct cell types in parallel. By mixing ES cell lines expressing myoD1 or the neural transcription factor ngn2, respectively, we generated a mixed culture of myocytes and neurons. Our findings provide new insights in the role of key developmental genes during cell fate decisions. Furthermore, this study represents an interesting strategy to obtain mixed cultures of different cells from stem cells, suggesting a valuable tool for cellular development and cell-cell interaction studies.

  18. Creation of Mice Bearing a Partial Duplication of HPRT Gene Marked with a GFP Gene and Detection of Revertant Cells In Situ as GFP-Positive Somatic Cells.

    Directory of Open Access Journals (Sweden)

    Asao Noda

    Full Text Available It is becoming clear that apparently normal somatic cells accumulate mutations. Such accumulations or propagations of mutant cells are thought to be related to certain diseases such as cancer. To better understand the nature of somatic mutations, we developed a mouse model that enables in vivo detection of rare genetically altered cells via GFP positive cells. The mouse model carries a partial duplication of 3' portion of X-chromosomal HPRT gene and a GFP gene at the end of the last exon. In addition, although HPRT gene expression was thought ubiquitous, the expression level was found insufficient in vivo to make the revertant cells detectable by GFP positivity. To overcome the problem, we replaced the natural HPRT-gene promoter with a CAG promoter. In such animals, termed HPRT-dup-GFP mouse, losing one duplicated segment by crossover between the two sister chromatids or within a single molecule of DNA reactivates gene function, producing hybrid HPRT-GFP proteins which, in turn, cause the revertant cells to be detected as GFP-positive cells in various tissues. Frequencies of green mutant cells were measured using fixed and frozen sections (liver and pancreas, fixed whole mount (small intestine, or by means of flow cytometry (unfixed splenocytes. The results showed that the frequencies varied extensively among individuals as well as among tissues. X-ray exposure (3 Gy increased the frequency moderately (~2 times in the liver and small intestine. Further, in two animals out of 278 examined, some solid tissues showed too many GFP-positive cells to score (termed extreme jackpot mutation. Present results illustrated a complex nature of somatic mutations occurring in vivo. While the HPRT-dup-GFP mouse may have a potential for detecting tissue-specific environmental mutagens, large inter-individual variations of mutant cell frequency cause the results unstable and hence have to be reduced. This future challenge will likely involve lowering the

  19. Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

    Directory of Open Access Journals (Sweden)

    Takahashi Takashi

    2007-04-01

    Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

  20. Macrodissection versus microdissection of rectal carcinoma: minor influence of stroma cells to tumor cell gene expression profiles

    Directory of Open Access Journals (Sweden)

    Medema Jan

    2005-10-01

    Full Text Available Abstract Background The molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor cell-specific gene expression profiles of these samples. To circumvent this complication, laser microdissection can be performed to separate tumor epithelium from the surrounding stroma and healthy tissue. In this report, we compared RNAs isolated from macrodissected, of which only surrounding healthy tissue had been removed, and microdissected rectal carcinoma samples by microarray analysis in order to determine the most reliable approach to detect the expression of tumor cell-derived genes by microarray analysis. Results As microdissection yielded low tissue and RNA quantities, extra rounds of mRNA amplification were necessary to obtain sufficient RNA for microarray experiments. These second rounds of amplification influenced the gene expression profiles. Moreover, the presence of stroma cells in macrodissected samples had a minor contribution to the tumor cell gene expression profiles, which can be explained by the observation that more RNA is extracted from tumor epithelial cells than from stroma. Conclusion These data demonstrate that the more convenient procedure of macrodissection can be adequately used and yields reliable data regarding the identification of tumor cell-specific gene expression profiles.

  1. HER4 selectively coregulates estrogen stimulated genes associated with breast tumor cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Han, Wen; Jones, Frank E., E-mail: fjones3@tulane.edu

    2014-01-10

    Highlights: •HER4/4ICD is an obligate coactivator for 37% of estrogen regulated genes. •HER4/4ICD coactivated genes selectively regulate estrogen stimulated proliferation. •Estrogen stimulated tumor cell migration occurs independent of HER4/4ICD. •Disrupting HER4/4ICD and ER coactivated gene expression may suppress breast cancer. -- Abstract: The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex is unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of β-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, β-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the β-estradiol stimulated genes. Ingenuity Pathway Analysis of β-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was

  2. Insulin but Not Glucagon Gene is Silenced in Human Pancreas-Derived Mesenchymal Stem Cells

    OpenAIRE

    2009-01-01

    We previously characterized human islet-derived precursor cells (hIPCs) as a specific type of mesenchymal stem cell capable of differentiating to insulin (INS)- and glucagon (GCG)-expressing cells. However, during proliferative expansion, INS transcript becomes undetectable and then cannot be induced, a phenomenon consistent with silencing of the INS gene. We explored this possibility by determining whether ectopic expression of transcription factors known to induce transcription of this gene...

  3. Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

    Science.gov (United States)

    Klein, U.; Klein, G.; Ehlin-Henriksson, B.; Rajewsky, K.; Küppers, R.

    1995-01-01

    BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher. PMID:8529116

  4. INHIBITION OF APOPTOSIS BY bcr-abl FUSION GENE IN K562 CELLS

    Institute of Scientific and Technical Information of China (English)

    WANG Chun-hong; SUN Bing-zhong; YUAN Yue-chuan

    1999-01-01

    Objective: To investigate the effect of bcr-abl fusion gene on CML cell apoptosis. Methods: Apoptosis of exvivo cultured K562 cells were observed after exposure to synthetic 18 mer antisense oligodeoxynucleotide complementary to the bcr-abl junction (b3a2). Results: Apoptosis of K562 cells was significantly increased associated with inhibition of bcr-abl expression. Conclusion: bcr-abl fusion gene formation due to chromosome translocation may be the major mechanism of CML via inhibition of apoptosis.

  5. Differential gene expression in CD45 cells at para-aortic foci stage of chicken haematopoiesis.

    Science.gov (United States)

    Säynäjäkangas, R; Uchida, T; Vainio, O

    2009-09-01

    Para-aortic foci of chicken embryos at 6-7 days of development are considered to provide a microenvironment for haematopoietic stem cell proliferation and initial differentiation similar to that of fetal liver in mammals. Here, we have investigated the genes involved in this process by constructing and analysing a subtractive cDNA library from CD45(+) cells in para-aortic region. Among 394 analysed clones 99 distinct genes were identified by sequence homology search. Classification of the identified genes according to biological processes revealed that innate immunity-related genes are highly expressed at this stage. This can be explained by the presence of yolk sac-derived macrophages in the original tissue sample but also by the indiscriminate expression of multiple lineage-specific genes in haematopoietic stem cells and primitive progenitors. Differentially expressed genes related to transcription, signalling and lymphocyte functions are potential candidates involved in lineage commitment.

  6. Expression of apoptosis-Related genes bcl-2 and bax in rat brain hippocampus, followed by intraperitoneal injection of nanosilver

    Directory of Open Access Journals (Sweden)

    Maryam Ghoshcian

    2016-05-01

    Full Text Available Background: Silver nanoparticles are small scale substance (<100 nm used in food technology and medical industry. The data suggest that nanosilver may produce neurotoxicity by generating free radical-induced oxidative stress and by altering gene expression producing apoptosis and neurotoxicity. In this study, the apoptotic effects of Nano silver on apoptosis- related genes expression bcl-2 and bax on rat hippocampus, which is involved in memory and learning, was investigated. Materials & Methods: 28 male Wistar rats were divided into four groups of control and three groups of the treatment. The control group received saline and the treatment groups received intraperitoneal injections of silver nanoparticles at doses of 100, 200 and 400ppm. Ten days after the last injection, the hippocampal region was dissected and removed and then the expression of bcl-2 and bax genes was evaluated using semi-qualitative RT-PCR and Densitometry assay. Results: The expression of anti- apoptotic b-cl2 gene was reduced in the treatment groups compared to the control group. In comparison, the expression of pro- apoptotic bax gene was increased in the treatment groups compared to the control group. This apoptotic affects was increased at higher doses. Conclusion: The data suggest that silver nanoparticles may produce apoptosis by altering apoptosis- related genes expression, in rat brain hippocampus cells.

  7. Clinicopathology, immunophenotype, T cell receptor gene rearrangement, Epstein-Barr virus status and p53 gene mutation of cutaneous extranodal NK/T-cell lymphoma, nasal-type

    Institute of Scientific and Technical Information of China (English)

    WANG Ting-ting; XU Chen; LIU Shan-ling; KAN Bei; RAN Yu-ping; LIU Wei-ping; LI Gan-di

    2013-01-01

    Background Extranodal natural killer/T-cell (NK/T cell) lymphoma,nasal-type,is a rare lymphoma.Skin is the second most common site of involvement after the nasal cavity/nasalpharynx.The aim of this study was to investigate the clinicopathologic features,immunophenotype,T cell receptor (TCR) gene rearrangement,the association with Epstein-Barr virus (EBV) infection and p53 gene mutations of the lymphoma.Methods The clinicopathologic analysis,immunohistochemistry,in situ hybridization for EBER1/2,TCR gene rearrangement by polymerase chain reaction (PCR),mutations of p53 gene analyzed by PCR and sequence analysis were employed in this study.Results In the 19 cases,the tumor primarily involved the dermis and subcutaneous layer.Immunohistochemical staining showed that most of the cases expressed CD45RO,CD56,CD3ε,TIA-1 and GrB.Three cases were positive for CD3 and two cases were positive for CD30.Monoclonal TCRY gene rearrangement was found in 7 of 18 cases.The positive rate of EBER1/2 was 100%.No p53 gene mutation was detected on the exon 4-9 in the 18 cases.Fifteen cases showed Pro (proline)/Arg (arginine) single nucleotide polymorphisms (SNPs) on the exon 4 at codon 72.The expression of p53 protein was 72% (13/18) immunohistochemically.Conclusions Cutaneous NK/T-cell lymphoma is a rare but highly aggressive lymphoma with poor prognosis.No p53 gene mutation was detected on the exon 4-9,and Pro/Arg SNPs on p53 codon 72 were detected in the cutaneous NK/T-cell lymphoma.The overexpression of p53 protein may not be the result of p53 gene mutation.

  8. Effects of hydrogen peroxide on mitochondrial gene expression of intestinal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Jian-Ming Li; Qian Cai; Hong Zhou; Guang-Xia Xiao

    2002-01-01

    AIM: To study the effects of hydrogen peroxide on mitochondrial gene expression of intestinal epithelial cells in in vitro model of hydrogen peroxide-stimulated SW-480 cells.METHODS: RNA of hydrogen peroxide-induced SW-480 cells was isolated, and reverse-transcriptional polymerase chain reaction was performed to study gene expression of ATPase subunit 6, ATPase subunit 8, cytochrome c oxidase subunit Ⅰ (COⅠ), cytochrome coxidase subuit Ⅱ (COⅡ) and cytochrome c oxidase subunit Ⅲ (COⅢ). Mitochondria were isolated and activities of mitochondrial cytochrome c oxidase and ATPase were also measured simultaneously.RESULTS: Hydrogen peroxide led to differential expression of mitochondrial genes with some genes up-regulated or down-regulated in a dose dependent manner. Differences were very obvious in expressions of mitochondrial genes of cells treated with hydrogen peroxide in a concentration of 400 μmol/L or 4 mmol/L. In general, differential expression of mitochondrial genes was characterized by up-regulation of mitochondrial genes in the concentration of 400 μmol/L and down-regulation in the concentration of 4 mmol/L. In consistence with changes in mitochondrial gene expressions, hydrogen peroxide resulted in decreased activities of cytochrome c oxidase and ATPase.CONCLUSIONS: The differential expression of mitochondrial genes encoding cytochrome c oxidase and ATPase is involved in apoptosis of intestinal epithelial cells by affecting activities of cytochorme c oxidase and ATPase.

  9. TNF-alpha-induced metastasis gene changes in MCF-7 cells

    Institute of Scientific and Technical Information of China (English)

    Xiaofeng Chen; Yongqian Shu; Wei Li; Yongmei Yin

    2008-01-01

    Objective: Studies have shown that TNF- a secreted by tumor cells and macrophages infiltrated into the tumor microenvironment might promote the metastasis of a variety of malignant cancers, including breast cancer. The present study was designed to detect global metastasis-related gene expression changes of MCF-7 cells treated by low dose TNF-a and to further explore the mechanisms by which TNF-a contributes to metastasis. Methods: MCF-7 cells were cultured and treated with low dose TNF-a (20 ng/ml), cDNA array analysis was applied to detect the metastasis related gene expressions. Results: A total of 36 gene expressions were significantly regulated by TNF-a. Functional analysis indicates that the altered genes belong to different functional group. Most of the genes changed may promote the metastasis of MCF-7 cells while the others may inhibit metastasis. The changes observed in gene expression following TNF-a were somewhat time dependent. Conclusion: TNF-a can enhance the invasive ability of MCF-7 cells, partly by regulating a series of metastasis related genes, and these genes may take part in every step of metastasis. Some of the genes deserve further study.

  10. Lineage-Specific Genes Are Prominent DNA Damage Hotspots during Leukemic Transformation of B Cell Precursors

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    Bryant Boulianne

    2017-02-01

    Full Text Available In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia.

  11. Expression Profile of Metastasis-associated Genes in Esophageal Squamous Cell Carcinoma

    Institute of Scientific and Technical Information of China (English)

    LI Pei; LING Zhiqiang; YANG Hongyan; HUANG Youtian; ZHAO Mingyao; ZHENG Zhimin; DONG Ziming

    2006-01-01

    The differentially expressed genes between esophageal squamous cell carcinoma (ESCC)with or without lymphatic metastasis were investigated by gene chip, and the lymphatic metastasisassociated genes were screened out. Expression array was used to detect the mRNA from both the primary carcinoma and the corresponding esophageal epithelium in 15 cases of human ESCC. The lymphatic metastasis-associated genes were screened by bioinformatics between ESCC with or without lymphatic metastasis. The results showed that 43 (4.85%) genes significantly differed between the ESCC with and without lymphatic metastasis (P<0.05), of which 18(2.03%)were upregulated and 25 (2.82 %) down-regulated. The up-regulated genes were involved in cell adhesion molecules and cell membrane receptors and the down-regulated genes were mostly cell cycle regulators and intracellular signaling molecules. It was suggested that lymphatic metastasis-associated genes were screened by gene chip, which was helpful to understand the molecular mechanism of ESCC lymphatic metastasis and lymphatic metastasis-associated genes might be used as diagnostic markers and therapeutic targets for lymphatic metastasis.

  12. Gene expression profiles in BCL11B-siRNA treated malignant T cells

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    Grabarczyk Piotr

    2011-05-01

    Full Text Available Abstract Background Downregulation of the B-cell chronic lymphocytic leukemia (CLL/lymphoma11B (BCL11B gene by small interfering RNA (siRNA leads to growth inhibition and apoptosis of the human T-cell acute lymphoblastic leukemia (T-ALL cell line Molt-4. To further characterize the molecular mechanism, a global gene expression profile of BCL11B-siRNA -treated Molt-4 cells was established. The expression profiles of several genes were further validated in the BCL11B-siRNA -treated Molt-4 cells and primary T-ALL cells. Results 142 genes were found to be upregulated and 109 genes downregulated in the BCL11B-siRNA -treated Molt-4 cells by microarray analysis. Among apoptosis-related genes, three pro-apoptotic genes, TNFSF10, BIK, BNIP3, were upregulated and one anti-apoptotic gene, BCL2L1 was downregulated. Moreover, the expression of SPP1 and CREBBP genes involved in the transforming growth factor (TGF-β pathway was down 16-fold. Expression levels of TNFSF10, BCL2L1, SPP1, and CREBBP were also examined by real-time PCR. A similar expression pattern of TNFSF10, BCL2L1, and SPP1 was identified. However, CREBBP was not downregulated in the BLC11B-siRNA -treated Molt-4 cells. Conclusion BCL11B-siRNA treatment altered expression profiles of TNFSF10, BCL2L1, and SPP1 in both Molt-4 T cell line and primary T-ALL cells.

  13. Identification of NDRG1-regulated genes associated with invasive potential in cervical and ovarian cancer cells

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    Zhao, Gang, E-mail: zhaog69@sjtu.edu.cn [Department of Pathology, The First People' s Hospital, Shanghai Jiaotong University, Shanghai (China); Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin (China); Chen, Jiawei, E-mail: jiaweichen2000@gmail.com [Department of Pathology, The First People' s Hospital, Shanghai Jiaotong University, Shanghai (China); Deng, Yanqiu [Pathophysiology Department, Tianjin Medical University, Tianjin (China); Gao, Feng [Department of Pathology, The First People' s Hospital, Shanghai Jiaotong University, Shanghai (China); Zhu, Jiwei [Basic Medical College, Harbin Medical University, Harbin (China); Feng, Zhenzhong; Lv, Xiuhong [Department of Pathology, The First People' s Hospital, Shanghai Jiaotong University, Shanghai (China); Zhao, Zheng [SAS Headquarters, S6013, 600 Research Drive, Cary, NC (United States)

    2011-04-29

    Highlights: {yields} NDRG1 was knockdown in cervical and ovarian cancer cell lines by shRNA technology. {yields} NDRG1 knockdown resulted in increased cell invasion activities. {yields} Ninety-six common deregulated genes in both cell lines were identified by cDNA microarray. {yields} Eleven common NDRG1-regulated genes might enhance cell invasive activity. {yields} Regulation of invasion by NDRG1 is an indirect and complicated process. -- Abstract: N-myc downstream regulated gene 1 (NDRG1) is an important gene regulating tumor invasion. In this study, shRNA technology was used to suppress NDRG1 expression in CaSki (a cervical cancer cell line) and HO-8910PM (an ovarian cancer cell line). In vitro assays showed that NDRG1 knockdown enhanced tumor cell adhesion, migration and invasion activities without affecting cell proliferation. cDNA microarray analysis revealed 96 deregulated genes with more than 2-fold changes in both cell lines after NDRG1 knockdown. Ten common upregulated genes (LPXN, DDR2, COL6A1, IL6, IL8, FYN, PTP4A3, PAPPA, ETV5 and CYGB) and one common downregulated gene (CLCA2) were considered to enhance tumor cell invasive activity. BisoGenet network analysis indicated that NDRG1 regulated these invasion effector genes/proteins in an indirect manner. Moreover, NDRG1 knockdown also reduced pro-invasion genes expression such as MMP7, TMPRSS4 and CTSK. These results suggest that regulation of invasion and metastasis by NDRG1 is a highly complicated process.

  14. Laser-based microdissection of single cells from tissue sections and PCR analysis of rearranged immunoglobulin genes from isolated normal and malignant human B cells.

    Science.gov (United States)

    Küppers, Ralf; Schneider, Markus; Hansmann, Martin-Leo

    2013-01-01

    Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene family-specific primers recognizing nearly all V gene segments together with primers for the J gene segments. By sequence analysis of V genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.

  15. High expression of hTERT and stemness genes in BORIS/CTCFL positive cells isolated from embryonic cancer cells.

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    Loredana Alberti

    Full Text Available BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3-5% of total. The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2 and cancer stem cell marker genes (CD44 and ALDH1 compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.

  16. Expression of Mesenchymal Stem Cells-Related Genes and Plasticity of Aspirated Follicular Cells Obtained from Infertile Women

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    Edo Dzafic

    2014-01-01

    Full Text Available After removal of oocytes for in vitro fertilization, follicular aspirates which are rich in somatic follicular cells are discarded in daily medical practice. However, there is some evidence that less differentiated cells with stem cell characteristics are present among aspirated follicular cells (AFCs. The aim of this study was to culture AFCs in vitro and to analyze their gene expression profile. Using the RT2 Profiler PCR array, we investigated the expression profile of 84 genes related to stemness, mesenchymal stem cells (MCSs, and cell differentiation in AFCs enriched by hypoosmotic protocol from follicular aspirates of infertile women involved in assisted reproduction programme in comparison with bone marrow-derived mesenchymal stem cells (BM-MSCs and fibroblasts. Altogether the expression of 57 genes was detected in AFCs: 16 genes (OCT4, CD49f, CD106, CD146, CD45, CD54, IL10, IL1B, TNF, VEGF, VWF, HDAC1, MITF, RUNX2, PPARG, and PCAF were upregulated and 20 genes (FGF2, CASP3, CD105, CD13, CD340, CD73, CD90, KDR, PDGFRB, BDNF, COL1A1, IL6, MMP2, NES, NUDT6, BMP6, SMURF2, BMP4, GDF5, and JAG1 were downregulated in AFCs when compared with BM-MSCs. The genes which were upregulated in AFCs were mostly related to MSCs and connected with ovarian function, and differed from those in fibroblasts. The cultured AFCs with predominating granulosa cells were successfully in vitro differentiated into adipogenic-, osteogenic-, and pancreatic-like cells. The upregulation of some MSC-specific genes and in vitro differentiation into other types of cells indicated a subpopulation of AFCs with specific stemness, which was not similar to those of BM-MSCs or fibroblasts.

  17. Different Effects of BORIS/CTCFL on Stemness Gene Expression, Sphere Formation and Cell Survival in Epithelial Cancer Stem Cells.

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    Loredana Alberti

    Full Text Available Cancer stem cells are cancer cells characterized by stem cell properties and represent a small population of tumor cells that drives tumor development, progression, metastasis and drug resistance. To date, the molecular mechanisms that generate and regulate cancer stem cells are not well defined. BORIS (Brother of Regulator of Imprinted Sites or CTCFL (CTCF-like is a DNA-binding protein that is expressed in normal tissues only in germ cells and is re-activated in tumors. Recent evidences have highlighted the correlation of BORIS/CTCFL expression with poor overall survival of different cancer patients. We have previously shown an association of BORIS-expressing cells with stemness gene expression in embryonic cancer cells. Here, we studied the role of BORIS in epithelial tumor cells. Using BORIS-molecular beacon that was already validated, we were able to show the presence of BORIS mRNA in cancer stem cell-enriched populations (side population and spheres of cervical, colon and breast tumor cells. BORIS silencing studies showed a decrease of sphere formation capacity in breast and colon tumor cells. Importantly, BORIS-silencing led to down-regulation of hTERT, stem cell (NANOG, OCT4, SOX2 and BMI1 and cancer stem cell markers (ABCG2, CD44 and ALDH1 genes. Conversely, BORIS-induction led to up-regulation of the same genes. These phenotypes were observed in cervical, colon and invasive breast tumor cells. However, a completely different behavior was observed in the non-invasive breast tumor cells (MCF7. Indeed, these cells acquired an epithelial mesenchymal transition phenotype after BORIS silencing. Our results demonstrate that BORIS is associated with cancer stem cell-enriched populations of several epithelial tumor cells and the different phenotypes depend on the origin of tumor cells.

  18. Macrodissection versus microdissection of rectal carcinoma : minor influence of stroma cells to tumor cell gene expression profiles

    NARCIS (Netherlands)

    Bruin, E.C. de; Pas, S. van de; Lips, E.H.; Eijk, R. van; Zee, M.M. van der; Lombaerts, M.; Wezel, T. van; Marijnen, C.A.M.; Krieken, J.H.J.M. van; Medema, J.P.; Velde, C.J. van de; Eilers, P.H.; Peltenburg, L.T.

    2005-01-01

    BACKGROUND: The molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor

  19. Study on interleukin-18 gene transfer into human breast cancer cells to prevent tumorigenicity

    Institute of Scientific and Technical Information of China (English)

    韩明勇; 郑树; 于金明; 彭佳萍; 郭其森; 王家林

    2004-01-01

    To study the effect of interleukin-18 gene transfection on the tumorigenesis of breast cancer cell line Bacp37, human breast cancer cell line Bcap37 were transfected with Lipofectamine and selected by G418. The biological expression of rhIL-18 was tested by RT-PCR and ELISA method; nude mice were injected with Bcap37 cell with or without the hIL-18 gene. The hIL-18 cDNA was successfully integrated into Bcap37 cell; 126.3±4.5 pg hIL-18 secreted by one million transduced cells in 24 hours. Nude mice injected with IL-18 gene engineered Bcap37 cell had no tumor growth. These findings indicated that human breast cancer cells were successfully modified by the gene of IL-18 cytokine; the IL-18 gene engineered Bcap37 cells secreted hIL-18 and lost their tumorigenicity. The Bcap37 cells transduced with IL-18 gene may be used as breast cancer vaccine.

  20. Study on interleukin-18 gene transfer into human breast cancer cells to prevent tumorigenicity

    Institute of Scientific and Technical Information of China (English)

    韩明勇; 郑树; 于金明; 彭佳萍; 郭其森; 王家林

    2004-01-01

    To study the effect of interleukin-18 gene transfection on the tumorigenesis of breast cancer cell line Bacp37,human breast cancer cell line Bcap37 were transfected with Lipofectamine and selected by G418.The biological expression of rhIL-18 was tested by RT-PCR and ELISA method;nude mice were injected with Bcap37 cell with or without the hIL-18 gene.The hIL-18 cDNA was successfully integrated into Bcap37 cell; 126.3±4.5pg hIL-18 secreted by one million transduced cells in 24 hours. Nude mice injected with IL-18 gene engineered Bcap37 cell had no tumor growth.These findings indicated that human breast cancer cells were successfully modified by the gene of IL-18 cytokine;the IL-18 gene engineered Bcap37 cells secreted hIL-18 and lost their tumorigenicity.The Bcap37 cells transduced with IL-18 gene may be used as breast cancer vaccine.

  1. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

  2. ADHESION INDUCES MATRIX METALLOPROTEINASE-9 GENE EXPRESSION IN OVARIAN CANCER CELLS

    Institute of Scientific and Technical Information of China (English)

    田方; 颜春洪; 薛红; 肖凤君

    2002-01-01

    Objective: To investigate the expression of matrix metalloproteinase-9 (MMP-9) gene in cancer cells induced by adhesion with fibronectin and the underlying mechanism of cell invasion. Methods: Following adhesion of ovarian cancer cells A2780 to fibronectin, MMP mRNA expression was assayed by using reverse transcription-polymerase chain reaction (RT-PCR). MMP-9 promoter was cloned from genomic DNA of HT1080 cells with PCR. The MMP-9-pGL2 reporter gene vector was constructed and then transiently transfected into A2780 cells. Results: Adhesion could induce the expression of MMP-9 gene in A2780 cells, but did not affect longer theexpression of MMP-2 or TIMP-1 gene. The induction was enhanced with longer adhesion time. When the transfected cells were allowed to adhere and spread on FN-coated surface, the promoter activity of MMP-9 gene was also enhanced dramatically. Conclusion: adhesion of cells with ECM may stimulate the expression of MMP-9 gene through stimulating the promoter activity, thereby enhancing cancer cell invasion and metastasis.

  3. GENE EXPRESSION CHANGES AFTER SEIZURE PRECONDITIONING IN THE THREE MAJOR HIPPOCAMPAL CELL LAYERS

    Science.gov (United States)

    Borges, Karin; Shaw, Renee; Dingledine, Raymond

    2008-01-01

    Rodents experience hippocampal damage after status epilepticus (SE) mainly in pyramidal cells while sparing the dentate granule cell layer (DGCL). Hippocampal damage was prevented in rats that had been preconditioned by brief seizures on two consecutive days before SE. To identify neuroprotective genes and biochemical pathways changed after preconditioning we compared the effect of preconditioning on gene expression in the CA1 and CA3 pyramidal and DGCLs, harvested by laser capture microscopy. In the DGCL the expression of 632 genes was altered, compared to only 151 and 58 genes in CA1 and CA3 pyramidal cell layers. Most of the differentially expressed genes regulate tissue structure and intra- and extracellular signaling, including neurotransmission. A selective upregulation of energy metabolism transcripts occurred in CA1 pyramidal cells relative to the DGCL. These results reveal a broad transcriptional response of the DGCL to preconditioning, and suggest several mechanisms underlying the neuroprotective effect of preconditioning seizures. PMID:17239605

  4. Rapid and Cost-Effective Gene Targeting in Rat Embryonic Stem Cells by TALENs

    Institute of Scientific and Technical Information of China (English)

    Chang Tong; Guanyi Huang; Charles Ashton; Hongping Wu; Hexin Yan; Qi-Long Ying

    2012-01-01

    The rat is the preferred animal model in many areas of biomedical research and drug development.Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools.Previously,we generated a knockout rat via conventional homolog