A New Approach to Establish a Cell Line with Reduced Risk of Endogenous Retroviruses

PubMed Central

Fukuma, Aiko; Yoshikawa, Rokusuke; Miyazawa, Takayuki; Yasuda, Jiro

2013-01-01

Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells). The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV. PMID:23585909

A new approach to establish a cell line with reduced risk of endogenous retroviruses.

PubMed

Fukuma, Aiko; Yoshikawa, Rokusuke; Miyazawa, Takayuki; Yasuda, Jiro

2013-01-01

Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells). The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV.

Functional and molecular evidence for expression of the renin angiotensin system and ADAM17-mediated ACE2 shedding in COS7 cells

PubMed Central

Grobe, Nadja; Di Fulvio, Mauricio; Kashkari, Nada; Chodavarapu, Harshita; Somineni, Hari K.; Singh, Richa

2015-01-01

The renin angiotensin system (RAS) plays a vital role in the regulation of the cardiovascular and renal functions. COS7 is a robust and easily transfectable cell line derived from the kidney of the African green monkey, Cercopithecus aethiops. The aims of this study were to 1) demonstrate the presence of an endogenous and functional RAS in COS7, and 2) investigate the role of a disintegrin and metalloproteinase-17 (ADAM17) in the ectodomain shedding of angiotensin converting enzyme-2 (ACE2). Reverse transcription coupled to gene-specific polymerase chain reaction demonstrated expression of ACE, ACE2, angiotensin II type 1 receptor (AT1R), and renin at the transcript levels in total RNA cell extracts. Western blot and immunohistochemistry identified ACE (60 kDa), ACE2 (75 kDa), AT1R (43 kDa), renin (41 kDa), and ADAM17 (130 kDa) in COS7. At the functional level, a sensitive and selective mass spectrometric approach detected endogenous renin, ACE, and ACE2 activities. ANG-(1–7) formation (m/z 899) from the natural substrate ANG II (m/z 1,046) was detected in lysates and media. COS7 cells stably expressing shRNA constructs directed against endogenous ADAM17 showed reduced ACE2 shedding into the media. This is the first study demonstrating endogenous expression of the RAS and ADAM17 in the widely used COS7 cell line and its utility to study ectodomain shedding of ACE2 mediated by ADAM17 in vitro. The transfectable nature of this cell line makes it an attractive cell model for studying the molecular, functional, and pharmacological properties of the renal RAS. PMID:25740155

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

PubMed

Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

2014-08-01

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.

PubMed

Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop

2015-01-01

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

PubMed

Stec, Wojciech J; Rosiak, Kamila; Siejka, Paulina; Peciak, Joanna; Popeda, Marta; Banaszczyk, Mateusz; Pawlowska, Roza; Treda, Cezary; Hulas-Bigoszewska, Krystyna; Piaskowski, Sylwester; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

2016-05-31

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII.The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes

PubMed Central

Stec, Wojciech J.; Rosiak, Kamila; Siejka, Paulina; Peciak, Joanna; Popeda, Marta; Banaszczyk, Mateusz; Pawlowska, Roza; Treda, Cezary; Hulas-Bigoszewska, Krystyna; Piaskowski, Sylwester; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

2016-01-01

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII. The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach. PMID:27004406

Role of Endogenous Cholecystokinin on Growth of Human Pancreatic Cancer

PubMed Central

Matters, Gail L.; McGovern, Christopher; Harms, John F.; Markovic, Kevin; Anson, Krystal; Jayakumar, Calpurnia; Martenis, Melissa; Awad, Christina; Smith, Jill P.

2012-01-01

Cholecystokinin (CCK) and gastrin stimulate growth of pancreatic cancer. Although down regulation of gastrin inhibits growth of pancreatic cancer, the contribution of endogenous CCK to tumor growth is unknown. The purpose of this study was to evaluate the role of endogenous CCK on autocrine growth of pancreatic cancer. Pancreatic cancer cell lines were analyzed for CCK mRNA and peptide expression by real time RT-PCR and radioimmunoassay, respectively. The effect of endogenous CCK on growth was evaluated by treating cancer cells with CCK neutralizing antibodies and by down regulating CCK mRNA by RNAi. Wild type pancreatic cancer cells expressed significantly lower CCK mRNA and peptide levels than gastrin. Neither treatment of pancreatic cancer cells with CCK antibodies nor the down regulation of CCK mRNA and peptide by shRNAs altered growth in vitro or in vivo. Conversely, when gastrin mRNA expression was down regulated, the same cells failed to produce tumors in spite of having sustained levels of endogenous CCK. Pancreatic cancer cells produce CCK and gastrin; however, the autocrine production of gastrin is more important for stimulating tumor growth. PMID:21186400

Utilization of the Tango beta-arrestin recruitment technology for cell-based EDG receptor assay development and interrogation.

PubMed

Wetter, Justin A; Revankar, Chetana; Hanson, Bonnie J

2009-10-01

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

PubMed

Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25 µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed. Copyright © 2011 Wiley Periodicals, Inc.

Conservative site-specific and single-copy transgenesis in human LINE-1 elements

PubMed Central

Vijaya Chandra, Shree Harsha; Makhija, Harshyaa; Peter, Sabrina; Myint Wai, Cho Mar; Li, Jinming; Zhu, Jindong; Ren, Zhonglu; D'Alcontres, Martina Stagno; Siau, Jia Wei; Chee, Sharon; Ghadessy, Farid John; Dröge, Peter

2016-01-01

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes. PMID:26673710

LncRNA HOTAIR acts a competing endogenous RNA to control the expression of notch3 via sponging miR-613 in pancreatic cancer.

PubMed

Cai, Huihua; Yao, Jie; An, Yong; Chen, Xuemin; Chen, Weibo; Wu, Di; Luo, Boyang; Yang, Yong; Jiang, Yong; Sun, Donglin; He, Xiaozhou

2017-05-16

Pancreatic cancer is one of the most deadly cancers with a poor prognosis. Though studies have implicated the roles of microRNAs in pancreatic cancer progression, little is known about the role of miR-613 in pancreatic cancer. In the present study, the expression of miR-613 was down-regulated in pancreatic cancer tissues and cancer cell lines. Down-regulation of miR-613 was positively correlated with tumor differentiation, advanced TNM stage, nodal metastasis and shorter overall survival in patients with pancreatic cancer. Overexpression of miR-613 suppressed cell proliferation, invasion and migration, and induced cell apoptosis and cell cycle arrest at G0/G1 phase in pancreatic cancer cells. Bioinformatics analysis, luciferase reporter assay and rescue experiments showed that notch3 was a direct target of miR-613. MiR-613 was inversely correlated with notch3 expression in pancreatic cancer tissues. The long non-coding RNA, HOX transcript antisense RNA (HOTAIR) was up-regulated in both pancreatic cancer tissues and cancer cell lines, and HOTAIR suppressed the expression of miR-613 via functioning as a competing endogenous RNA. In vivo studies showed that stable overexpression of miR-613 or knock-down of HOTAIR suppressed tumor growth and also reduced the expression of notch3. In conclusion, these results suggest that HOTAIR functions as a competing endogenous RNA to regulate notch3 expression via sponging miR-613 in pancreatic cancer.

Arabidopsis PIZZA has the capacity to acylate brassinosteroids.

PubMed

Schneider, Katja; Breuer, Christian; Kawamura, Ayako; Jikumaru, Yusuke; Hanada, Atsushi; Fujioka, Shozo; Ichikawa, Takanari; Kondou, Youichi; Matsui, Minami; Kamiya, Yuji; Yamaguchi, Shinjiro; Sugimoto, Keiko

2012-01-01

Brassinosteroids (BRs) affect a wide range of developmental processes in plants and compromised production or signalling of BRs causes severe growth defects. To identify new regulators of plant organ growth, we searched the Arabidopsis FOX (Full-length cDNA Over-eXpressor gene) collection for mutants with altered organ size and isolated two overexpression lines that display typical BR deficient dwarf phenotypes. The phenotype of these lines, caused by an overexpression of a putative acyltransferase gene PIZZA (PIZ), was partly rescued by supplying exogenous brassinolide (BL) and castasterone (CS), indicating that endogenous BR levels are rate-limiting for the growth of PIZ overexpression lines. Our transcript analysis further showed that PIZ overexpression leads to an elevated expression of genes involved in BR biosynthesis and a reduced expression of BR inactivating hydroxylases, a transcriptional response typical to low BR levels. Taking the advantage of relatively high endogenous BR accumulation in a mild bri1-301 background, we found that overexpression of PIZ results in moderately reduced levels of BL and CS and a strong reduction of typhasterol (TY) and 6-deoxocastasterone (6-deoxoCS), suggesting a role of PIZ in BR metabolism. We tested a set of potential substrates in vitro for heterologously expressed PIZ and confirmed its acyltransferase activity with BL, CS and TY. The PIZ gene is expressed in various tissues but as reported for other genes involved in BR metabolism, the loss-of-function mutants did not display obvious growth phenotypes under standard growth conditions. Together, our data suggest that PIZ can modify BRs by acylation and that these properties might help modulating endogenous BR levels in Arabidopsis.

Differential Expression of HERV-K (HML-2) Proviruses in Cells and Virions of the Teratocarcinoma Cell Line Tera-1

PubMed Central

Bhardwaj, Neeru; Montesion, Meagan; Roy, Farrah; Coffin, John M.

2015-01-01

Human endogenous retrovirus (HERV-K (HML-2)) proviruses are among the few endogenous retroviral elements in the human genome that retain coding sequence. HML-2 expression has been widely associated with human disease states, including different types of cancers as well as with HIV-1 infection. Understanding of the potential impact of this expression requires that it be annotated at the proviral level. Here, we utilized the high throughput capabilities of next-generation sequencing to profile HML-2 expression at the level of individual proviruses and secreted virions in the teratocarcinoma cell line Tera-1. We identified well-defined expression patterns, with transcripts emanating primarily from two proviruses located on chromosome 22, only one of which was efficiently packaged. Interestingly, there was a preference for transcripts of recently integrated proviruses, over those from other highly expressed but older elements, to be packaged into virions. We also assessed the promoter competence of the 5’ long terminal repeats (LTRs) of expressed proviruses via a luciferase assay following transfection of Tera-1 cells. Consistent with the RNASeq results, we found that the activity of most LTRs corresponded to their transcript levels. PMID:25746218

Mechanisms of allele-selective down-regulation of HLA class I in Burkitt's lymphoma.

PubMed

Imreh, M P; Zhang, Q J; de Campos-Lima, P O; Imreh, S; Krausa, P; Browning, M; Klein, G; Masucci, M G

1995-07-04

Burkitt lymphomas (BL) that arise in HLA-AII-positive individuals are characterized by selective loss/down-regulation of the HLA AII polypeptide. We have investigated the molecular basis of such down-regulation by comparing 5 pairs of BL lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL) derived from the normal B cells of the same individuals. The presence of apparently intact HLA AII genes was confirmed in all 5 BL/LCL pairs by polymerase chain reaction (PCR) typing and by Southern-blot hybridization with HLA A locus-specific probes. Northern-blot analysis with locus- and allele-specific probes revealed a significantly lower expression or absence of AII-specific mRNA in all 5 BL lines compared to the corresponding LCLs. Up-regulation of AII-specific mRNA was achieved by IFN alpha treatment of 2 BL lines with low HLA AII expression (BL-28 and BL-72) while the treatment had no effect in 3 BL lines (WWI-BL, WW2-BL and BL41) that did not express the endogenous gene. HLA AII expression was restored by transfection of the gene in WWI-BL whereas transfectants of BL-41 remained AII-negative. An HLA-AII-promoter-driven chloramphenicol acetyl transferase reporter gene (pAIICAT) was active in WWI-BL but not in BL-41. HLA-AII was expressed in hybrids of BL-41 with an AII-positive LCL, while expression of the endogenous HLA AII gene could not be restored by fusion of BL-41 with an AII-negative LCL, although an adequate set of transcription factors was present in the hybrid. Our results suggest that genetic defects and lack of transcription factors may contribute to the selective down-regulation of HLA AII in BL cells.

Murine Endogenous Retroviruses Are Detectable in Patient-Derived Xenografts but Not in Patient-Individual Cell Lines of Human Colorectal Cancer.

PubMed

Bock, Stephanie; Mullins, Christina S; Klar, Ernst; Pérot, Philippe; Maletzki, Claudia; Linnebacher, Michael

2018-01-01

Endogenous retroviruses are remnants of retroviral infections. In contrast to their human counterparts, murine endogenous retroviruses (mERV) still can synthesize infectious particles and retrotranspose. Xenotransplanted human cells have occasionally been described to be mERV infected. With genetic engineered mice and patient-derived xenografts (PDXs) on the rise as eminent research tools, we here systematically investigated, if different tumor models harbor mERV infections. Relevant mERV candidates were first preselected by next generation sequencing (NGS) analysis of spontaneous lymphomas triggered by colorectal cancer (CRC) PDX tissue. Two primer systems were designed for each of these candidates (AblMLV, EcoMLV, EndoPP, MLV, and preXMRV) and implemented in an quantitative real-time (RT-qPCR) screen using murine tissues ( n = 11), PDX-tissues ( n = 22), PDX-derived cell lines ( n = 13), and patient-derived tumor cell lines ( n = 14). The expression levels of mERV varied largely both in the PDX samples and in the mouse tissues. No mERV signal was, however, obtained from cDNA or genomic DNA of CRC cell lines. Expression of EcoMLV was higher in PDX than in murine tissues; for EndoPP it was the opposite. These two were thus further investigated in 40 additional PDX. In addition, four patient-derived cell lines free of any mERV expression were subcutaneously injected into immunodeficient mice. Outgrowing cell-derived xenografts barely expressed EndoPP. In contrast, the expression of EcoMLV was even higher than in surrounding mouse tissues. This expression gradually vanished within few passages of re-cultivated cells. In summary, these results strongly imply that: (i) PDX and murine tissues in general are likely to be contaminated by mERV, (ii) mERV are expressed transiently and at low level in fresh PDX-derived cell cultures, and (iii) mERV integration into the genome of human cells is unlikely or at least a very rare event. Thus, mERVs are stowaways present in murine cells, in PDX tissues and early thereof-derived cell cultures. We conclude that further analysis is needed concerning their impact on results obtained from studies performed with PDX but also with murine tumor models.

The role of heterologous chloroplast sequence elements in transgene integration and expression.

PubMed

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-04-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression1[W][OA

PubMed Central

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-01-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5′ untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5′ UTR and 3′ UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5′ UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5′ UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation. PMID:20130101

The endogenous and reactive depression subtypes revisited: integrative animal and human studies implicate multiple distinct molecular mechanisms underlying major depressive disorder.

PubMed

Malki, Karim; Keers, Robert; Tosto, Maria Grazia; Lourdusamy, Anbarasu; Carboni, Lucia; Domenici, Enrico; Uher, Rudolf; McGuffin, Peter; Schalkwyk, Leonard C

2014-05-07

Traditional diagnoses of major depressive disorder (MDD) suggested that the presence or absence of stress prior to onset results in either 'reactive' or 'endogenous' subtypes of the disorder, respectively. Several lines of research suggest that the biological underpinnings of 'reactive' or 'endogenous' subtypes may also differ, resulting in differential response to treatment. We investigated this hypothesis by comparing the gene-expression profiles of three animal models of 'reactive' and 'endogenous' depression. We then translated these findings to clinical samples using a human post-mortem mRNA study. Affymetrix mouse whole-genome oligonucleotide arrays were used to measure gene expression from hippocampal tissues of 144 mice from the Genome-based Therapeutic Drugs for Depression (GENDEP) project. The study used four inbred mouse strains and two depressogenic 'stress' protocols (maternal separation and Unpredictable Chronic Mild Stress) to model 'reactive' depression. Stress-related mRNA differences in mouse were compared with a parallel mRNA study using Flinders Sensitive and Resistant rat lines as a model of 'endogenous' depression. Convergent genes differentially expressed across the animal studies were used to inform candidate gene selection in a human mRNA post-mortem case control study from the Stanley Brain Consortium. In the mouse 'reactive' model, the expression of 350 genes changed in response to early stresses and 370 in response to late stresses. A minimal genetic overlap (less than 8.8%) was detected in response to both stress protocols, but 30% of these genes (21) were also differentially regulated in the 'endogenous' rat study. This overlap is significantly greater than expected by chance. The VAMP-2 gene, differentially expressed across the rodent studies, was also significantly altered in the human study after correcting for multiple testing. Our results suggest that 'endogenous' and 'reactive' subtypes of depression are associated with largely distinct changes in gene-expression. However, they also suggest that the molecular signature of 'reactive' depression caused by early stressors differs considerably from that of 'reactive' depression caused by late stressors. A small set of genes was consistently dysregulated across each paradigm and in post-mortem brain tissue of depressed patients suggesting a final common pathway to the disorder. These genes included the VAMP-2 gene, which has previously been associated with Axis-I disorders including MDD, bipolar depression, schizophrenia and with antidepressant treatment response. We also discuss the implications of our findings for disease classification, personalized medicine and case-control studies of MDD.

Cellular Prion Protein Combined with Galectin-3 and -6 Affects the Infectivity Titer of an Endogenous Retrovirus Assayed in Hippocampal Neuronal Cells

PubMed Central

Shin, Hae-Young; Goto, Joy J.; Carp, Richard I.; Choi, Eun-Kyoung; Kim, Yong-Sun

2016-01-01

Prion diseases are infectious and fatal neurodegenerative diseases which require the cellular prion protein, PrPC, for development of diseases. The current study shows that the PrPC augments infectivity and plaque formation of a mouse endogenous retrovirus, MuLV. We have established four neuronal cell lines expressing mouse PrPC, PrP+/+; two express wild type PrPC (MoPrPwild) and the other two express mutant PrPC (MoPrPmut). Infection of neuronal cells from various PrP+/+ and PrP-/- (MoPrPKO) lines with MuLV yielded at least three times as many plaques in PrP+/+ than in PrP-/-. Furthermore, among the four PrP+/+ lines, one mutant line, P101L, had at least 2.5 times as many plaques as the other three PrP+/+ lines. Plaques in P101L were four times larger than those in other PrP+/+ lines. Colocalization of PrP and CAgag was seen in MuLV-infected PrP+/+ cells. In the PrP-MuLV interaction, the involvement of galectin-3 and -6 was observed by immunoprecipitation with antibody to PrPC. These results suggest that PrPC combined with galectin-3 and -6 can act as a receptor for MuLV. P101L, the disease form of mutant PrPC results suggest the genetic mutant form of PrPC may be more susceptible to viral infection. PMID:27936017

Erythroid Kruppel-like factor (EKLF) is recruited to the γ-globin gene promoter as a co-activator and is required for γ-globin gene induction by short-chain fatty acid derivatives

PubMed Central

Perrine, Susan P.; Mankidy, Rishikesh; Boosalis, Michael S.; Bieker, James J.; Faller, Douglas V.

2011-01-01

Objectives The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for β-type globin gene switching, and specifically activates transcription of the adult β-globin gene promoter. We sought to determine if EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. Methods The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Results and conclusions Knockdown of EKLF prevents SCFA-induced expression of the γ-globin promoter in a stably expressed μLCRβprRlucAγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous γ-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for γ-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous γ-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. PMID:19220418

Hili Inhibits HIV Replication in Activated T Cells.

PubMed

Peterlin, B Matija; Liu, Pingyang; Wang, Xiaoyun; Cary, Daniele; Shao, Wei; Leoz, Marie; Hong, Tian; Pan, Tao; Fujinaga, Koh

2017-06-01

P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4 + T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNA Arg (UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements. IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4 + T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements. Copyright © 2017 American Society for Microbiology.

An Aryl Hydrocarbon Receptor-Mediated Amplification Loop That Enforces Cell Migration in ER-/PR-/Her2- Human Breast Cancer Cells.

PubMed

Novikov, Olga; Wang, Zhongyan; Stanford, Elizabeth A; Parks, Ashley J; Ramirez-Cardenas, Alejandra; Landesman, Esther; Laklouk, Israa; Sarita-Reyes, Carmen; Gusenleitner, Daniel; Li, Amy; Monti, Stefano; Manteiga, Sara; Lee, Kyongbum; Sherr, David H

2016-11-01

The endogenous ligand-activated aryl hydrocarbon receptor (AHR) plays an important role in numerous biologic processes. As the known number of AHR-mediated processes grows, so too does the importance of determining what endogenous AHR ligands are produced, how their production is regulated, and what biologic consequences ensue. Consequently, our studies were designed primarily to determine whether ER - /PR - /Her2 - breast cancer cells have the potential to produce endogenous AHR ligands and, if so, how production of these ligands is controlled. We postulated that: 1) malignant cells produce tryptophan-derived AHR ligand(s) through the kynurenine pathway; 2) these metabolites have the potential to drive AHR-dependent breast cancer migration; 3) the AHR controls expression of a rate-limiting kynurenine pathway enzyme(s) in a closed amplification loop; and 4) environmental AHR ligands mimic the effects of endogenous ligands. Data presented in this work indicate that primary human breast cancers, and their metastases, express high levels of AHR and tryptophan-2,3-dioxygenase (TDO); representative ER - /PR - /Her2 - cell lines express TDO and produce sufficient intracellular kynurenine and xanthurenic acid concentrations to chronically activate the AHR. TDO overexpression, or excess kynurenine or xanthurenic acid, accelerates migration in an AHR-dependent fashion. Environmental AHR ligands 2,3,7,8-tetrachlorodibenzo[p]dioxin and benzo[a]pyrene mimic this effect. AHR knockdown or inhibition significantly reduces TDO2 expression. These studies identify, for the first time, a positive amplification loop in which AHR-dependent TDO2 expression contributes to endogenous AHR ligand production. The net biologic effect of AHR activation by endogenous ligands, which can be mimicked by environmental ligands, is an increase in tumor cell migration, a measure of tumor aggressiveness. Copyright © 2016 by The Author(s).

An Aryl Hydrocarbon Receptor-Mediated Amplification Loop That Enforces Cell Migration in ER−/PR−/Her2− Human Breast Cancer Cells

PubMed Central

Novikov, Olga; Wang, Zhongyan; Stanford, Elizabeth A.; Parks, Ashley J.; Ramirez-Cardenas, Alejandra; Landesman, Esther; Laklouk, Israa; Sarita-Reyes, Carmen; Gusenleitner, Daniel; Li, Amy; Monti, Stefano; Manteiga, Sara; Lee, Kyongbum

2016-01-01

The endogenous ligand-activated aryl hydrocarbon receptor (AHR) plays an important role in numerous biologic processes. As the known number of AHR-mediated processes grows, so too does the importance of determining what endogenous AHR ligands are produced, how their production is regulated, and what biologic consequences ensue. Consequently, our studies were designed primarily to determine whether ER−/PR−/Her2− breast cancer cells have the potential to produce endogenous AHR ligands and, if so, how production of these ligands is controlled. We postulated that: 1) malignant cells produce tryptophan-derived AHR ligand(s) through the kynurenine pathway; 2) these metabolites have the potential to drive AHR-dependent breast cancer migration; 3) the AHR controls expression of a rate-limiting kynurenine pathway enzyme(s) in a closed amplification loop; and 4) environmental AHR ligands mimic the effects of endogenous ligands. Data presented in this work indicate that primary human breast cancers, and their metastases, express high levels of AHR and tryptophan-2,3-dioxygenase (TDO); representative ER−/PR−/Her2− cell lines express TDO and produce sufficient intracellular kynurenine and xanthurenic acid concentrations to chronically activate the AHR. TDO overexpression, or excess kynurenine or xanthurenic acid, accelerates migration in an AHR-dependent fashion. Environmental AHR ligands 2,3,7,8-tetrachlorodibenzo[p]dioxin and benzo[a]pyrene mimic this effect. AHR knockdown or inhibition significantly reduces TDO2 expression. These studies identify, for the first time, a positive amplification loop in which AHR-dependent TDO2 expression contributes to endogenous AHR ligand production. The net biologic effect of AHR activation by endogenous ligands, which can be mimicked by environmental ligands, is an increase in tumor cell migration, a measure of tumor aggressiveness. PMID:27573671

Endogenous T cell responses to antigens expressed in lung adenocarcinomas delay malignant tumor progression

PubMed Central

DuPage, Michel; Cheung, Ann; Mazumdar, Claire; Winslow, Monte M.; Bronson, Roderick; Schmidt, Leah M.; Crowley, Denise; Chen, Jianzhu; Jacks, Tyler

2010-01-01

SUMMARY Neoantigens derived from somatic mutations in tumors may provide a critical link between the adaptive immune system and cancer. Here we describe a system to introduce exogenous antigens into genetically engineered mouse lung cancers to mimic tumor neoantigens. We show that endogenous T cells respond to and infiltrate tumors, significantly delaying malignant progression. Despite continued antigen expression, T cell infiltration does not persist and tumors ultimately escape immune attack. Transplantation of cell lines derived from these lung tumors or prophylactic vaccination against the autochthonous tumors, however, results in rapid tumor eradication or selection of tumors that lose antigen expression. These results provide insight into the dynamic nature of the immune response to naturally arising tumors. PMID:21251614

Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors

PubMed Central

Sacco, Joseph J.; Kenyani, Jenna; Butt, Zohra; Carter, Rachel; Chew, Hui Yi; Cheeseman, Liam P.; Darling, Sarah; Denny, Michael; Urbé, Sylvie; Clague, Michael J.; Coulson, Judy M.

2015-01-01

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors. PMID:25970771

CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development

PubMed Central

Kesavan, Gokul; Chekuru, Avinash; Machate, Anja; Brand, Michael

2017-01-01

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a. The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development. PMID:28713249

CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development.

PubMed

Kesavan, Gokul; Chekuru, Avinash; Machate, Anja; Brand, Michael

2017-01-01

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a . The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development.

Melatonin-induced CBF/DREB1s are essential for diurnal change of disease resistance and CCA1 expression in Arabidopsis.

PubMed

Shi, Haitao; Wei, Yunxie; He, Chaozu

2016-03-01

Melatonin (N-acetyl-5-methoxytryptamine) is an important regulator of circadian rhythms and immunity in animals. However, the diurnal changes of endogenous melatonin and melatonin-mediated diurnal change of downstream responses remain unclear in Arabidopsis. Using the publicly available microarray data, we found that the transcript levels of two melatonin synthesis genes (serotonin N-acetyltransferase (SNAT) and caffeate O-methyltransferase (COMT)) and endogenous melatonin level were regulated by diurnal cycles, with different magnitudes of change. Moreover, the transcripts of C-repeat-binding factors (CBFs)/Drought response element Binding 1 factors (DREB1s) were co-regulated by exogenous melatonin and diurnal changes, indicating the possible correlation among clock, endogenous melatonin level and AtCBFs expressions. Interestingly, diurnal change of plant immunity against Pst DC3000 and CIRCADIANCLOCK ASSOCIATED 1 (CCA1) expression were largely lost in AtCBFs knockdown line-amiR-1. Taken together, this study identifies the molecular pathway underlying the diurnal changes of immunity in Arabidopsis. Notably, the diurnal changes of endogenous melatonin may regulate corresponding changes of AtCBF/DREB1s expression and their underlying diurnal cycle of plant immunity and AtCCA1. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

PubMed

Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

2002-08-01

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

Transfection of cultured cells of the cotton boll weevil, Anthonomus grandis, with a heat-shock-promoter-chloramphenicol-acetyltransferase construct.

PubMed

Stiles, B; Heilmann, J; Sparks, R B; Santoso, A; Leopold, R A

1992-01-01

Expression of heat shock proteins (hsp) in the BRL-AG-3C cell line from the cotton boll weevil was examined. It was determined that the maximal expression of endogenous hsp occurred at 41 degrees C. Various transfection methods were then compared using this cell line in conjunction with a transiently expressed bacterial gene marker (chloramphenicol acetyltransferase) which was under the control of the Drosophila hsp 70 gene promoter. The cationic lipid preparation Lipofectin was found to be very efficient at transfecting the boll weevil cells. Polylysine and 20-hydroxyecdysone-conjugated polylysine were moderately effective, whereas polybrene and electroporation, under the conditions reported herein, were ineffective at transfecting this cell line.

Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

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

Cheng, Xiaoxia; University of Chinese Academy of Sciences, Beijing; Chen, Xiaowen

The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were allmore » identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.« less

Increasing the yield of middle silk gland expression system through transgenic knock-down of endogenous sericin-1.

PubMed

Ma, Sanyuan; Xia, Xiaojuan; Li, Yufeng; Sun, Le; Liu, Yue; Liu, Yuanyuan; Wang, Xiaogang; Shi, Run; Chang, Jiasong; Zhao, Ping; Xia, Qingyou

2017-08-01

Various genetically modified bioreactor systems have been developed to meet the increasing demands of recombinant proteins. Silk gland of Bombyx mori holds great potential to be a cost-effective bioreactor for commercial-scale production of recombinant proteins. However, the actual yields of proteins obtained from the current silk gland expression systems are too low for the proteins to be dissolved and purified in a large scale. Here, we proposed a strategy that reducing endogenous sericin proteins would increase the expression yield of foreign proteins. Using transgenic RNA interference, we successfully reduced the expression of BmSer1 to 50%. A total 26 transgenic lines expressing Discosoma sp. red fluorescent protein (DsRed) in the middle silk gland (MSG) under the control of BmSer1 promoter were established to analyze the expression of recombinant. qRT-PCR and western blotting showed that in BmSer1 knock-down lines, the expression of DsRed had significantly increased both at mRNA and protein levels. We did an additional analysis of DsRed/BmSer1 distribution in cocoon and effect of DsRed protein accumulation on the silk fiber formation process. This study describes not only a novel method to enhance recombinant protein expression in MSG bioreactor, but also a strategy to optimize other bioreactor systems.

Role of Crk Adaptor Proteins in Cellular Migration and Invasion in Human Breast Cancer

DTIC Science & Technology

2008-03-01

interact with the endogenous CrkL binding partner, Gab1 , which is enhanced upon HGF stimulation. The over- expression of CrkL and Gab1 leads to Rac...transgenic line in both virgin and lactating mice (Figure 1C). Moreover, although the antibody recognizes endogenous protein, it is clearly evident that...found equal levels in both the nucleus and cytoplasm using two different antibodies targeting CrkI/II or specifically CrkII (7). Furthermore, we have

Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride

PubMed Central

Brütting, Christine; Narasimhan, Harini; Hoffmann, Frank; Kornhuber, Malte E.; Staege, Martin S.; Emmer, Alexander

2018-01-01

Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS. PMID:29515560

Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride.

PubMed

Brütting, Christine; Narasimhan, Harini; Hoffmann, Frank; Kornhuber, Malte E; Staege, Martin S; Emmer, Alexander

2018-01-01

Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS.

Nuclear hormone retinoid X receptor (RXR) negatively regulates the glucose-stimulated insulin secretion of pancreatic ß-cells.

PubMed

Miyazaki, Satsuki; Taniguchi, Hidenori; Moritoh, Yusuke; Tashiro, Fumi; Yamamoto, Tsunehiko; Yamato, Eiji; Ikegami, Hiroshi; Ozato, Keiko; Miyazaki, Jun-ichi

2010-11-01

Retinoid X receptors (RXRs) are members of the nuclear hormone receptor superfamily and are thought to be key regulators in differentiation, cellular growth, and gene expression. Although several experiments using pancreatic β-cell lines have shown that the ligands of nuclear hormone receptors modulate insulin secretion, it is not clear whether RXRs have any role in insulin secretion. To elucidate the function of RXRs in pancreatic β-cells, we generated a double-transgenic mouse in which a dominant-negative form of RXRβ was inducibly expressed in pancreatic β-cells using the Tet-On system. We also established a pancreatic β-cell line from an insulinoma caused by the β-cell-specific expression of simian virus 40 T antigen in the above transgenic mouse. In the transgenic mouse, expression of the dominant-negative RXR enhanced the insulin secretion with high glucose stimulation. In the pancreatic β-cell line, the suppression of RXRs also enhanced glucose-stimulated insulin secretion at a high glucose concentration, while 9-cis-retinoic acid, an RXR agonist, repressed it. High-density oligonucleotide microarray analysis showed that expression of the dominant-negative RXR affected the expression levels of a number of genes, some of which have been implicated in the function and/or differentiation of β-cells. These results suggest that endogenous RXR negatively regulates the glucose-stimulated insulin secretion. Given these findings, we propose that the modulation of endogenous RXR in β-cells may be a new therapeutic approach for improving impaired insulin secretion in type 2 diabetes.

Gibberellin 3-oxidase Gene Expression Patterns Influence Gibberellin Biosynthesis, Growth, and Development in Pea1[W][OPEN

PubMed Central

Reinecke, Dennis M.; Wickramarathna, Aruna D.; Ozga, Jocelyn A.; Kurepin, Leonid V.; Jin, Alena L.; Good, Allen G.; Pharis, Richard P.

2013-01-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth. PMID:23979969

Gibberellin 3-oxidase gene expression patterns influence gibberellin biosynthesis, growth, and development in pea.

PubMed

Reinecke, Dennis M; Wickramarathna, Aruna D; Ozga, Jocelyn A; Kurepin, Leonid V; Jin, Alena L; Good, Allen G; Pharis, Richard P

2013-10-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.

Down-regulation of human endogenous retrovirus type K (HERV-K) viral env RNA in pancreatic cancer cells decreases cell proliferation and tumor growth

PubMed Central

Li, Ming; Radvanyi, Laszlo; Yin, Bingnan; Li, Jia; Chivukula, Raghavender; Lin, Kevin; Lu, Yue; Shen, JianJun; Chang, David Z.; Li, Donghui; Johanning, Gary L.; Wang-Johanning, Feng

2017-01-01

Purpose We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer (PC). Experimental Design shRNA was employed to knockdown (KD) the expression of HERV-K in PC cells. Results HERV-K env expression was detected in seven PC cell lines and in 80% of PC patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several PC cell lines. RT activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in PC patient sera (N=106) than in normal donor sera (N=40). Importantly, the in vitro and in vivo growth rates of three PC cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several PC cells or tumors. Conclusion These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in PC. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of PC, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis and immunotherapy of PC. PMID:28679769

Epigenetic Alterations Associated With CCCTC-Binding Factor Deregulation in Prostate Cancer

DTIC Science & Technology

2011-07-01

HPV16 E6 and/or E7 prostate cell lines. We have established stable cell lines containing inducible CTCF shRNA in pTRIPZ vector in PPC-1, LNCaPs, 293T...and non-tumorigenic HPV16 E6 and/or E7 prostate cell lines. We are in process of conducting CTCF knockdown experiments using transient transfection...which express high levels of endogenous CTCF and in non- tumorigenic HPV16 E6 and/or E7 prostate cell lines. We see efficient knockdown of CTCF

Cell line specific modulation of connexin43 expression after exposure to ionizing radiation.

PubMed

Banaz-Yaşar, Ferya; Tischka, Rabea; Iliakis, George; Winterhager, Elke; Gellhaus, Alexandra

2005-01-01

Gap junctional intercellular communication plays a significant role in mediating radiation-induced bystander effects. However, the level of Cx43 itself is influenced by ionizing radiation, which could modify the bystander effect. Here we have investigated several cell lines for the modulation of Cx43 expression 24 h after irradiation with 5 Gy X-rays. The mouse endothelial cell line bEnd3 revealed a significantly elevated level of Cx43 already 15 min after exposure to X-rays, whereas human hybrid endothelial cells (EA.hy926) exhibited a transient downregulation of Cx43 mRNA. No obvious changes in the communication properties of the different cell lines could be observed after irradiation. The communication-deficient malignant human trophoblast cell line Jeg3 stably transfected with Cx43 did not reveal any induction of endogenous nor alteration in the exogenous Cx43 transcript level upon exposure to 5 Gy. Taken together, our data show a cell line specific modulation of Cx43 expression after exposure to X-rays.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

PubMed

Novosadova, E V; Manuilova, E S; Arsen'eva, E L; Khaidarova, N V; Dolotov, O V; Inozemtseva, L S; Kozachenkov, K Yu; Tarantul, V Z; Grivennikov, I A

2005-07-01

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

Human Mas-Related G Protein-Coupled Receptors-X1 Induce Chemokine Receptor 2 Expression in Rat Dorsal Root Ganglia Neurons and Release of Chemokine Ligand 2 from the Human LAD-2 Mast Cell Line

PubMed Central

Solinski, Hans Jürgen; Petermann, Franziska; Rothe, Kathrin; Boekhoff, Ingrid; Gudermann, Thomas; Breit, Andreas

2013-01-01

Primate-specific Mas-related G protein-coupled receptors-X1 (MRGPR-X1) are highly enriched in dorsal root ganglia (DRG) neurons and induce acute pain. Herein, we analyzed effects of MRGPR-X1 on serum response factors (SRF) or nuclear factors of activated T cells (NFAT), which control expression of various markers of chronic pain. Using HEK293, DRG neuron-derived F11 cells and cultured rat DRG neurons recombinantly expressing human MRGPR-X1, we found activation of a SRF reporter gene construct and induction of the early growth response protein-1 via extracellular signal-regulated kinases-1/2 known to play a significant role in the development of inflammatory pain. Furthermore, we observed MRGPR-X1-induced up-regulation of the chemokine receptor 2 (CCR2) via NFAT, which is considered as a key event in the onset of neuropathic pain and, so far, has not yet been described for any endogenous neuropeptide. Up-regulation of CCR2 is often associated with increased release of its endogenous agonist chemokine ligand 2 (CCL2). We also found MRGPR-X1-promoted release of CCL2 in a human connective tissue mast cell line endogenously expressing MRGPR-X1. Thus, we provide first evidence to suggest that MRGPR-X1 induce expression of chronic pain markers in DRG neurons and propose a so far unidentified signaling circuit that enhances chemokine signaling by acting on two distinct yet functionally co-operating cell types. Given the important role of chemokine signaling in pain chronification, we propose that interruption of this signaling circuit might be a promising new strategy to alleviate chemokine-promoted pain. PMID:23505557

Endogenous protein "barcode" for data validation and normalization in quantitative MS analysis.

PubMed

Lee, Wooram; Lazar, Iulia M

2014-07-01

Quantitative proteomic experiments with mass spectrometry detection are typically conducted by using stable isotope labeling and label-free quantitation approaches. Proteins with housekeeping functions and stable expression level such actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase are frequently used as endogenous controls. Recent studies have shown that the expression level of such common housekeeping proteins is, in fact, dependent on various factors such as cell type, cell cycle, or disease status and can change in response to a biochemical stimulation. The interference of such phenomena can, therefore, substantially compromise their use for data validation, alter the interpretation of results, and lead to erroneous conclusions. In this work, we advance the concept of a protein "barcode" for data normalization and validation in quantitative proteomic experiments. The barcode comprises a novel set of proteins that was generated from cell cycle experiments performed with MCF7, an estrogen receptor positive breast cancer cell line, and MCF10A, a nontumorigenic immortalized breast cell line. The protein set was selected from a list of ~3700 proteins identified in different cellular subfractions and cell cycle stages of MCF7/MCF10A cells, based on the stability of spectral count data generated with an LTQ ion trap mass spectrometer. A total of 11 proteins qualified as endogenous standards for the nuclear and 62 for the cytoplasmic barcode, respectively. The validation of the protein sets was performed with a complementary SKBR3/Her2+ cell line.

Corticotropin-Releasing Hormone Modulates Human Trophoblast Invasion through Carcinoembryonic Antigen-Related Cell Adhesion Molecule-1 Regulation

PubMed Central

Bamberger, Ana-Maria; Minas, Vassilis; Kalantaridou, Sophia N.; Radde, Jessica; Sadeghian, Helen; Löning, Thomas; Charalampopoulos, Ioannis; Brümmer, Jens; Wagener, Christoph; Bamberger, Christoph M.; Schulte, Heinrich M.; Chrousos, George P.; Makrigiannakis, Antonis

2006-01-01

Abnormalities in the process of trophoblast invasion may result in abnormal placentation. Both the embryonic trophoblast and maternal decidua produce corticotropin-releasing hormone (CRH), which promotes implantation. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is expressed in extravillous trophoblasts (EVTs) of normal human placenta, may also function in tro-phoblast/endometrial interactions. We investigated whether locally produced CRH plays a role in trophoblast invasion, primarily by regulating CEACAM1 expression. We examined cultures of freshly isolated human EVTs, which express CEACAM1, and an EVT-based hybridoma cell line, which is devoid of endogenous CEACAM1. CRH inhibited EVT invasion in Matrigel invasion assays, and this effect was blocked by the CRH receptor type 1 (CRHR1)-specific antagonist antalarmin. Additionally, CRH decreased CEACAM1 expression in EVTs in a dose-dependent manner. After transfection of the hybridoma cell line with a CEACAM1 expression vector, the invasiveness of these cells was strongly enhanced. This effect was inhibited by addition of blocking monoclonal antibody against CEACAM1. Furthermore, blocking of endogenous CEACAM1 in EVTs inhibited the invasive potential of these cells. Taken together these findings suggest that CRH inhibits trophoblast invasion by decreasing the expression of CEACAM1 through CRHR1, an effect that might be involved in the pathophysiology of clinical conditions, such as preeclampsia and placenta accreta. PMID:16400017

Regulation of Histone Deacetylase 4 Expression by the SP Family of Transcription FactorsD⃞

PubMed Central

Liu, Fang; Pore, Nabendu; Kim, Mijin; Voong, K. Ranh; Dowling, Melissa; Maity, Amit; Kao, Gary D.

2006-01-01

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4. PMID:16280357

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

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

Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique

2013-04-01

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and themore » ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.« less

Reactivation of Embryonic Nodal Signaling is Associated with Tumor Progression and Promotes the Growth of Prostate Cancer Cells

PubMed Central

Lawrence, Mitchell G.; Margaryan, Naira V.; Loessner, Daniela; Collins, Angus; Kerr, Kris M.; Turner, Megan; Seftor, Elisabeth A.; Stephens, Carson R.; Lai, John; BioResource, APC; Postovit, Lynne-Marie; Clements, Judith A.; Hendrix, Mary J.C.

2011-01-01

Background Nodal is a member of the Transforming Growth Factor β (TGFβ) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFβ ligands, it modulates androgen receptor (AR) activity. Methods Nodal expression was investigated using immunohistochemistry of tissue microarrays and Western blots of prostate cell lines. The functional role of Nodal was examined using Matrigel and soft agar growth assays. Cross-talk between Nodal and AR signaling was assessed with luciferase reporter assays and expression of endogenous androgen regulated genes. Results Significantly increased Nodal expression was observed in cancer compared with benign prostate specimens. Nodal was only expressed by DU145 and PC3 cells. All cell lines expressed Nodal’s co-receptor, Cripto-1, but lacked Lefty, a critical negative regulator of Nodal signaling. Recombinant human Nodal triggered downstream Smad2 phosphorylation in DU145 and LNCaP cells, and stable transfection of pre-pro-Nodal enhanced the growth of LNCaP cells in Matrigel and soft agar. Finally, Nodal attenuated AR signaling, reducing the activity of a PSA promoter construct in luciferase assays and down-regulating the endogenous expression of androgen regulated genes. Conclusions An aberrant Nodal signaling pathway is re-expressed and functionally active in prostate cancer cells. PMID:21656830

Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

PubMed Central

Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G

1997-01-01

Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875

Butyrate enhances disease resistance of chickens by inducing antimicrobial host defense peptide gene expression

USDA-ARS?s Scientific Manuscript database

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. I...

MicroRNA-29b Regulates the Expression Level of Human Progranulin, a Secreted Glycoprotein Implicated in Frontotemporal Dementia

PubMed Central

Jiao, Jian; Herl, Lauren D.; Farese, Robert V.; Gao, Fen-Biao

2010-01-01

Progranulin deficiency is thought to cause some forms of frontotemporal dementia (FTD), a major early-onset age-dependent neurodegenerative disease. How progranulin (PGRN) expression is regulated is largely unknown. We identified an evolutionarily conserved binding site for microRNA-29b (miR-29b) in the 3′ untranslated region (3′UTR) of the human PGRN (hPGRN) mRNA. miR-29b downregulates the expression of luciferase through hPGRN or mouse PGRN (mPGRN) 3′UTRs, and the regulation was abolished by mutations in the miR-29b binding site. To examine the direct effect of manipulating endogenous miR-29b on hPGRN expression, we established a stable NIH3T3 cell line that expresses hPGRN under the control of the cytomegalovirus promoter. Ectopic expression of miR-29b decreased hPGRN expression at the both mRNA and protein levels. Conversely, knockdown of endogenous miR-29b with locked nucleic acid increased the production and secretion of hPGRN in NIH3T3 cells. Endogenous hPGRN in HEK 293 cells was also regulated by miR-29b. These findings identify miR-29b as a novel posttranscriptional regulator of PGRN expression, raising the possibility that miR-29b or other miRNAs might be targeted therapeutically to increase hPGRN levels in some FTD patients. PMID:20479936

The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

PubMed

Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

2011-02-01

In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

Regulator of G protein signaling 4 inhibits human melanoma cells proliferation and invasion through the PI3K/AKT signaling pathway

PubMed Central

Xue, Xiaotong; Wang, Lihua; Meng, Xianguang; Jiao, Jing; Dang, Ningning

2017-01-01

Melanoma is a tumor produced by skin melanocytes, which has a high metastatic rate and poor prognosis. So far, plenty of work has been done on melanoma, but mechanisms underlying melanoma development have not been fully elucidated. Here we identified regulator of G protein signaling 4(RGS4) as novel therapeutic target for malignant melanoma and its regulating effect on melanoma. We found that endogenous RGS4 expression was much lower in melanoma tissues and cells. In A375 cell line with low endogenous RGS4 expression, the function of RGS4 was detected by up-regulation its expression with pcDNA3.1-RGS4 and knockdown its expression with siRNA. Our results showed that RGS4 could significantly reduce the proliferation, migration and invasion of melanoma cells. RGS4 is an important regulator for the apoptosis of melanocyte, and the apoptosis rate is significantly decreased in low RGS4 enviroment. RGS4 induced non-activation of PI3K/AKT pathway, resulting in decreased expression of E2F1 and Cyclin D1, thus constraining cell proliferation and invasion. These results were further confirmed in M14 cell lines. Collectively, our findings show that RGS4 plays an important role in multiple cellular functions of melanoma development and is valuable to be a therapeutic target. PMID:29108247

Improving panicle exsertion of rice cytoplasmic male sterile line by combination of artificial microRNA and artificial target mimic.

PubMed

Chen, Hao; Jiang, Shan; Zheng, Jie; Lin, Yongjun

2013-04-01

The adoption of hybrid rice caused the second leap in rice yield after the 'green revolution' and contributes substantially to food security of China and the world. However, almost all cytoplasmic male sterile lines (A lines) as females of hybrid rice have a natural deficiency of 'panicle enclosure', which blocks pollination between the A line and the fertility restorer line as the male (R line) of hybrid rice and decreases seed yield. In hybrid rice seed production, exogenous '920' (the active ingredient is gibberellin A3 ) must be applied to eliminate or alleviate panicle enclosure of the A line; however, this not only increases production cost and pollutes the environment, it also decreases seed quality. In this study, we designed a transgenic approach to improve plant height and panicle exsertion of the A line to facilitate hybrid rice production and maintain the semi-dwarf plant type of the hybrid. This approach comprising two components-artificial microRNA (amiRNA) and artificial target mimicry-can manipulate the differential expression of the endogenous Eui1 gene that is associated with rice internode elongation in the A line and the hybrid. amiRNA is a recently developed gene silencing method with high specificity, while target mimicry is a natural mechanism inhibiting the miRNA function that was also recently characterized. This approach provides a paradigm to tune the expression of endogenous genes to achieve the desired phenotype by combining amiRNA and artificial target mimicry technologies. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Recent Amplification of the Kangaroo Endogenous Retrovirus, KERV, Limited to the Centromere▿

PubMed Central

Ferreri, Gianni C.; Brown, Judith D.; Obergfell, Craig; Jue, Nathaniel; Finn, Caitlin E.; O'Neill, Michael J.; O'Neill, Rachel J.

2011-01-01

Mammalian retrotransposons, transposable elements that are processed through an RNA intermediate, are categorized as short interspersed elements (SINEs), long interspersed elements (LINEs), and long terminal repeat (LTR) retroelements, which include endogenous retroviruses. The ability of transposable elements to autonomously amplify led to their initial characterization as selfish or junk DNA; however, it is now known that they may acquire specific cellular functions in a genome and are implicated in host defense mechanisms as well as in genome evolution. Interactions between classes of transposable elements may exert a markedly different and potentially more significant effect on a genome than interactions between members of a single class of transposable elements. We examined the genomic structure and evolution of the kangaroo endogenous retrovirus (KERV) in the marsupial genus Macropus. The complete proviral structure of the kangaroo endogenous retrovirus, phylogenetic relationship among relative retroviruses, and expression of this virus in both Macropus rufogriseus and M. eugenii are presented for the first time. In addition, we show the relative copy number and distribution of the kangaroo endogenous retrovirus in the Macropus genus. Our data indicate that amplification of the kangaroo endogenous retrovirus occurred in a lineage-specific fashion, is restricted to the centromeres, and is not correlated with LINE depletion. Finally, analysis of KERV long terminal repeat sequences using massively parallel sequencing indicates that the recent amplification in M. rufogriseus is likely due to duplications and concerted evolution rather than a high number of independent insertion events. PMID:21389136

Dexamethasone enhances interaction of endogenous annexin 1 with L-selectin and triggers shedding of L-selectin in the monocytic cell line U-937.

PubMed

de Coupade, Catherine; Solito, Egle; Levine, Jon D

2003-09-01

(1) L-selectin, constitutively expressed by leukocytes, is involved in the initial binding of leukocytes to activated endothelium. Anti-inflammatory drugs like glucocorticoids can induce shedding of L-selectin, but the mechanism is still unknown. Annexin 1, a protein whose synthesis and externalization/secretion are induced during the inflammatory response, has been proposed as a mediator of the anti-inflammatory actions of glucocorticoids. (2) The monocytic cell line U-937 strongly expresses Annexin 1 after 24 h of phorbol 12-myristate 13-acetate (PMA, 1 nm) treatment and externalizes/releases the protein after additional 16 h of dexamethasone (1 microm) treatment. (3) This study investigated the possible regulation of cell surface L-selectin shedding by endogenous Annexin 1, and its role in glucocorticoid-induced L-selectin shedding in the U-937 cell line. (4) PMA- and dexamethasone treatment-induced L-selectin shedding was potentially mediated by Annexin 1, since neutralizing antibodies against Annexin 1 reduced dexamethasone- and Annexin 1-induced shedding. (5) Immunoprecipitation and binding assays provided support for the suggestion that this effect could be mediated by an interaction between externalized Annexin 1 and L-selectin. Such interaction involved the N-terminal domain of Annexin 1 and was calcium-dependent. Confocal microscopy studies demonstrated increased colocalization of Annexin 1 and L-selectin on the cell surface. (6) Overall, our study provides new insights into the potential role of endogenous ANXA1 as a mediator of dexamethasone-induced L-selectin shedding, which may contribute to the anti-inflammatory activity of glucocorticoids.

Role of endogenous insulin gene enhancer protein ISL-1 in angiogenesis

PubMed Central

Xiong, Si-qi; Jiang, Hai-bo; Li, Yan-xiu; Li, Hai-bo; Xu, Hui-zhuo; Wu, Zhen-kai; Zheng, Wei

2016-01-01

Objective To elucidate the role of insulin gene enhancer protein ISL-1 (Islet-1) in angiogenesis and regulation of vascular endothelial growth factor (VEGF) expression in vitro and in vivo. Methods siRNA targeting Islet-1 was transfected to human umbilical vein endothelial cell lines (HUVECs). The expression of Islet-1 and VEGF in the cultured cells was measured using real-time PCR and immunoblotting. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to analyze the proliferation of HUVECs affected by Islet-1. Wound healing and Transwell assays were conducted to assess the motility of HUVECs. The formation of capillary-like structures was examined using growth factor–reduced Matrigel. siRNA targeting Islet-1 was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated with angiography using fluorescein-labeled dextran and then quantified histologically. Real-time PCR and immunoblotting were used to determine whether local Islet-1 silencing affected the expression of Islet-1 and VEGF in murine retinas. Results The expression of Islet-1 and VEGF in HUVECs was knocked down by siRNA. Reduced endogenous Islet-1 levels in cultured cells greatly inhibited the proliferation, migration, and tube formation in HUVECs in vitro. Retinal neovascularization following injection of Islet-1 siRNA was significantly reduced compared with that of the contralateral control eye. Histological analysis indicated that the neovascular nuclei protruding into the vitreous cavity were decreased. Furthermore, the Islet-1 and VEGF expression levels were downregulated in murine retinas treated with siRNA against Islet-1. Conclusions Reducing the expression of endogenous Islet-1 inhibits proliferation, migration, and tube formation in vascular endothelial cells in vitro and suppresses retinal angiogenesis in vivo. Endogenous Islet-1 regulates angiogenesis via VEGF. PMID:27994436

Concise classification of the genomic porcine endogenous retroviral gamma1 load to defined lineages.

PubMed

Klymiuk, Nikolai; Wolf, Eckhard; Aigner, Bernhard

2008-02-05

We investigated the infection history of porcine endogenous retroviruses (PERV) gamma1 by analyzing published env and LTR sequences. PERV sequences from various breeds, porcine cell lines and infected human primary cells were included in the study. We identified a considerable number of retroviral lineages indicating multiple independent colonization events of the porcine genome. A recent boost of the proviral load in an isolated pig herd and exclusive occurrence of distinct lineages in single studies indicated the ongoing colonization of the porcine genome with endogenous retroviruses. Retroviral recombination between co-packaged genomes was a general factor for PERV gamma1 diversity which indicated the simultaneous expression of different proviral loci over a period of time. In total, our detailed description of endogenous retroviral lineages is the prerequisite for breeding approaches to minimize the infectious potential of porcine tissues for the subsequent use in xenotransplantation.

Long noncoding RNA BDNF-AS is associated with clinical outcomes and has functional role in human prostate cancer.

PubMed

Li, Wensheng; Dou, Zhongling; We, Shuguang; Zhu, Zhiyi; Pan, Dong; Jia, Zhaohui; Liu, Hui; Wang, Xiaobin; Yu, Guoqiang

2018-06-01

The underlying molecular mechanisms of prostate cancer (CaP) are largely unknown. We investigated the expression, prognostic value and functional role of long non-coding RNA (lncRNA) brain-derived neurotrophin factor antisense (BDNF-AS) in CaP. Clinical tumor samples were excised from patients with CaP. Their endogenous BDNF-AS expression levels were evaluated by qRT-PCR. Correlations between CaP patients' endogenous BDNF-AS expression and their clinicopathological factors, overall survival were statistically analyzed. BDNF-AS expression levels were also probed in immortal CaP cell lines. In LNCaP and PC-3 cells, BDNF-AS was ectopically overexpressed through lentiviral transduction. The functions of BDNF-AS upregulation on CaP cell development were evaluated both in vitro and in vivo. BDNF-AS was downregulated in human CaP tumors. Low BDNF-AS expression was correlated with CaP patients' poor prognosis and shorter overall survival. BDNF-AS was also found to be lowly expressed in CaP cell lines. In LNCaP and PC-3 cells, lentivirus-driven BDNF-AS overexpression exerted significantly tumor-suppressing effects on hindering cancer cell proliferation and invasion in vitro, and explant growth in vivo. Downregulated BDNF-AS in CaP patients could be a potential prognostic biomarker for predicating poor prognosis and survival. Upregulating BDNF-AS may be a novel molecular intervening target for CaP treatment. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Establishing Substantial Equivalence: Transcriptomics

NASA Astrophysics Data System (ADS)

Baudo, María Marcela; Powers, Stephen J.; Mitchell, Rowan A. C.; Shewry, Peter R.

Regulatory authorities in Western Europe require transgenic crops to be substantially equivalent to conventionally bred forms if they are to be approved for commercial production. One way to establish substantial equivalence is to compare the transcript profiles of developing grain and other tissues of transgenic and conventionally bred lines, in order to identify any unintended effects of the transformation process. We present detailed protocols for transcriptomic comparisons of developing wheat grain and leaf material, and illustrate their use by reference to our own studies of lines transformed to express additional gluten protein genes controlled by their own endosperm-specific promoters. The results show that the transgenes present in these lines (which included those encoding marker genes) did not have any significant unpredicted effects on the expression of endogenous genes and that the transgenic plants were therefore substantially equivalent to the corresponding parental lines.

Transient SNAIL1 expression is necessary for metastatic competence in breast cancer.

PubMed

Tran, Hung D; Luitel, Krishna; Kim, Michael; Zhang, Kun; Longmore, Gregory D; Tran, David D

2014-11-01

SNAIL1 has been suggested to regulate breast cancer metastasis based on analyses of human breast tumor transcriptomes and experiments using cancer cell lines and xenografts. However, in vivo genetic experimental support for a role for SNAIL1 in breast cancer metastasis that develops in an immunocompetent tumor microenvironment has not been determined. To address this question, we created a genetic SNAIL1 model by coupling an endogenous SNAIL1 reporter with an inducible SNAIL1 transgene. Using multiple genetic models of breast cancer, we demonstrated that endogenous SNAIL1 expression was restricted to primary tumors that ultimately disseminate. SNAIL1 gene deletion either during the premalignant phase or after primary tumors have reached a palpable size blunted metastasis, indicating that late metastasis was the main driver of metastasis and that this was dependent on SNAIL1. Importantly, SNAIL1 expression during breast cancer metastasis was transient and forced transient, but not continuous. SNAIL1 expression in breast tumors was sufficient to increase metastasis. ©2014 American Association for Cancer Research.

Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

PubMed Central

Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

1991-01-01

Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

Nuclear location of an endogenously expressed antigen, EBNA1, restricts access to macroautophagy and the range of CD4 epitope display.

PubMed

Leung, Carol S; Haigh, Tracey A; Mackay, Laura K; Rickinson, Alan B; Taylor, Graham S

2010-02-02

Whereas exogenously acquired proteins are the major source of antigens feeding the MHC class II pathway in antigen-presenting cells, some endogenously expressed antigens also access that pathway but the rules governing such access are poorly understood. Here we address this using Epstein-Barr virus (EBV)-coded nuclear antigen EBNA1, a protein naturally expressed in EBV-infected B lymphoblastoid cell lines (LCLs) and a source of multiple CD4(+) T cell epitopes. Using CD4(+) T cell clones against three indicator epitopes, we find that two epitopes are weakly displayed on the LCL surface whereas the third is undetectable, a pattern of limited epitope presentation that is maintained even when nuclear expression of EBNA1 is induced to high supraphysiological levels. Inhibitor and siRNA studies show that, of the two epitopes weakly presented under these conditions, one involves macroautophagy, and the second involves antigen delivery to the MHC II pathway by another endogenous route. In contrast, when EBNA1 is expressed as a cytoplasmic protein, all three CD4 epitopes are processed and presented much more efficiently, and all involve macroautophagy. We conclude that EBNA1's nuclear location limits its accessibility to the macroautophagy pathway and, in consequence, limits the level and range of EBNA1 CD4 epitopes naturally displayed on the infected cell surface.

A novel positive transcriptional feedback loop in midbrain-hindbrain boundary development is revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines.

PubMed

Picker, Alexander; Scholpp, Steffen; Böhli, Heike; Takeda, Hiroyuki; Brand, Michael

2002-07-01

The pax2.1 gene encodes a paired-box transcription factor that is one of the earliest genes to be specifically activated in development of the midbrain and midbrain-hindbrain boundary (MHB), and is required for the development and organizer activity of this territory. To understand how this spatially restricted transcriptional activity of pax2.1 is achieved, we have isolated and characterized the pax2.1-promoter using a lacZ and a GFP reporter gene in transient injection assays and transgenic lines. Stable transgenic expression of this reporter gene shows that a 5.3-kb fragment of the 5' region contains most, but not all, elements required for driving pax2.1 expression. The expressing tissues include the MHB, hindbrain, spinal cord, ear and pronephros. Transgene activation in the pronephros and developing ear suggests that these pax2.1-expressing tissues are composed of independently regulated subdomains. In addition, ectopic but spatially restricted activation of the reporter genes in rhombomeres 3 and 5 and in the forebrain, which do not normally express endogenous pax2.1, demonstrates the importance of negative regulation of pax2.1. Comparison of transgene expression in wild-type and homozygous pax2.1 mutant no isthmus (noi) embryos reveals that the transgene contains control element(s) for a novel, positive transcriptional feedback loop in MHB development. Transcription of endogenous pax2.1 at the MHB is known to be initially Pax2.1 independent, during activation in late gastrulation. In contrast, transgene expression requires the endogenous Pax2.1 function. Transplantations, mRNA injections and morpholino knock-down experiments show that this feedback regulation of pax2.1 transcription occurs cell-autonomously, and that it requires eng2 and eng3 as known targets for Pax2.1 regulation. We suggest that this novel feedback loop may allow continuation of pax2.1 expression, and hence development of the MHB organizer, to become independent of the patterning machinery of the gastrula embryo.

Functional Dissection of the Neural Substrates for Sexual Behaviors in Drosophila melanogaster

PubMed Central

Meissner, Geoffrey W.; Manoli, Devanand S.; Chavez, Jose F.; Knapp, Jon-Michael; Lin, Tasha L.; Stevens, Robin J.; Mellert, David J.; Tran, David H.; Baker, Bruce S.

2011-01-01

The male-specific Fruitless proteins (FruM) act to establish the potential for male courtship behavior in Drosophila melanogaster and are expressed in small groups of neurons throughout the nervous system. We screened ∼1000 GAL4 lines, using assays for general courtship, male–male interactions, and male fertility to determine the phenotypes resulting from the GAL4-driven inhibition of FruM expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups on the basis of additional neurobiological and behavioral criteria. For example, in some lines, restoration of FruM expression in cholinergic neurons restores fertility or reduces male–male courtship. Persistent chains of males courting each other in some lines results from males courting both sexes indiscriminately, whereas in other lines this phenotype results from apparent habituation deficits. Inhibition of ectopic FruM expression in females, in populations of neurons where FruM is necessary for male fertility, can rescue female infertility. To identify the neurons responsible for some of the observed behavioral alterations, we determined the overlap between the identified GAL4 lines and endogenous FruM expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with FruM expression in many regions of the nervous system, suggesting likely redundant FruM-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation. PMID:21705753

Specific pesticide-dependent increases in α-synuclein levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines.

PubMed

Chorfa, Areski; Bétemps, Dominique; Morignat, Eric; Lazizzera, Corinne; Hogeveen, Kevin; Andrieu, Thibault; Baron, Thierry

2013-06-01

Epidemiological studies indicate a role of genetic and environmental factors in Parkinson's disease involving alterations of the neuronal α-synuclein (α-syn) protein. In particular, a relationship between Parkinson's disease and occupational exposure to pesticides has been repeatedly suggested. Our objective was to precisely assess changes in α-syn levels in human neuroblastoma (SH-SY5Y) and melanoma (SK-MEL-2) cell lines following acute exposure to pesticides (rotenone, paraquat, maneb, and glyphosate) using Western blot and flow cytometry. These human cell lines express α-syn endogenously, and overexpression of α-syn (wild type or mutated A53T) can be obtained following recombinant adenoviral transduction. We found that endogenous α-syn levels in the SH-SY5Y neuroblastoma cell line were markedly increased by paraquat, and to a lesser extent by rotenone and maneb, but not by glyphosate. Rotenone also clearly increased endogenous α-syn levels in the SK-MEL-2 melanoma cell line. In the SH-SY5Y cell line, similar differences were observed in the α-syn adenovirus-transduced cells, with a higher increase of the A53T mutated protein. Paraquat markedly increased α-syn in the SK-MEL-2 adenovirus-transduced cell line, similarly for the wild-type or A53T proteins. The observed differences in the propensities of pesticides to increase α-syn levels are in agreement with numerous reports that indicate a potential role of exposure to certain pesticides in the development of Parkinson's disease. Our data support the hypothesis that pesticides can trigger some molecular events involved in this disease and also in malignant melanoma that consistently shows a significant but still unexplained association with Parkinson's disease.

Upregulated LINE-1 Activity in the Fanconi Anemia Cancer Susceptibility Syndrome Leads to Spontaneous Pro-inflammatory Cytokine Production.

PubMed

Brégnard, Christelle; Guerra, Jessica; Déjardin, Stéphanie; Passalacqua, Frank; Benkirane, Monsef; Laguette, Nadine

2016-06-01

Fanconi Anemia (FA) is a genetic disorder characterized by elevated cancer susceptibility and pro-inflammatory cytokine production. Using SLX4(FANCP) deficiency as a working model, we questioned the trigger for chronic inflammation in FA. We found that absence of SLX4 caused cytoplasmic DNA accumulation, including sequences deriving from active Long INterspersed Element-1 (LINE-1), triggering the cGAS-STING pathway to elicit interferon (IFN) expression. In agreement, absence of SLX4 leads to upregulated LINE-1 retrotransposition. Importantly, similar results were obtained with the FANCD2 upstream activator of SLX4. Furthermore, treatment of FA cells with the Tenofovir reverse transcriptase inhibitor (RTi), that prevents endogenous retrotransposition, decreased both accumulation of cytoplasmic DNA and pro-inflammatory signaling. Collectively, our data suggest a contribution of endogenous RT activities to the generation of immunogenic cytoplasmic nucleic acids responsible for inflammation in FA. The additional observation that RTi decreased pro-inflammatory cytokine production induced by DNA replication stress-inducing drugs further demonstrates the contribution of endogenous RTs to sustaining chronic inflammation. Altogether, our data open perspectives in the prevention of adverse effects of chronic inflammation in tumorigenesis. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

PubMed

Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest

PubMed Central

Gascoyne, Duncan M.; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E.; Croucher, Peter I.; Banham, Alison H.

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology. PMID:26034982

Ferritin Assembly in Enterocytes of Drosophila melanogaster

PubMed Central

Rosas-Arellano, Abraham; Vásquez-Procopio, Johana; Gambis, Alexis; Blowes, Liisa M.; Steller, Hermann; Mollereau, Bertrand; Missirlis, Fanis

2016-01-01

Ferritins are protein nanocages that accumulate inside their cavity thousands of oxidized iron atoms bound to oxygen and phosphates. Both characteristic types of eukaryotic ferritin subunits are present in secreted ferritins from insects, but here dimers between Ferritin 1 Heavy Chain Homolog (Fer1HCH) and Ferritin 2 Light Chain Homolog (Fer2LCH) are further stabilized by disulfide-bridge in the 24-subunit complex. We addressed ferritin assembly and iron loading in vivo using novel transgenic strains of Drosophila melanogaster. We concentrated on the intestine, where the ferritin induction process can be controlled experimentally by dietary iron manipulation. We showed that the expression pattern of Fer2LCH-Gal4 lines recapitulated iron-dependent endogenous expression of the ferritin subunits and used these lines to drive expression from UAS-mCherry-Fer2LCH transgenes. We found that the Gal4-mediated induction of mCherry-Fer2LCH subunits was too slow to effectively introduce them into newly formed ferritin complexes. Endogenous Fer2LCH and Fer1HCH assembled and stored excess dietary iron, instead. In contrast, when flies were genetically manipulated to co-express Fer2LCH and mCherry-Fer2LCH simultaneously, both subunits were incorporated with Fer1HCH in iron-loaded ferritin complexes. Our study provides fresh evidence that, in insects, ferritin assembly and iron loading in vivo are tightly regulated. PMID:26861293

TGF-beta1 expression in EL4 lymphoma cells overexpressing growth hormone.

PubMed

Farmer, John T; Weigent, Douglas A

2006-03-01

Our previous studies show that growth hormone overexpression (GHo) upregulates the expression of the IGF-1R and IGF-2R resulting in the protection of the EL4 lymphoma cell line from apoptosis. In this study, we report that GHo also increases TGF-beta1 protein expression measured by luciferase promoter assay, Western analysis, and ELISA. Further, the data show that antibody to TGF-betaR2 decreases TGF-beta1 promoter activity to the level of vector alone control cells. GHo cells treated with (125)I-rh-latent TGF-beta1 showed increased activation of latent TGF-beta1 as measured by an increase in the active 24kDa, TGF-beta1 compared to vector alone control cells. The ability of endogenous GH to increase TGF-beta1 expression is blocked in EL4 cells by antisense but not sense oligodeoxynucleotides or in cells cultured with antibody to growth hormone (GH). The data suggest that endogenous GH may protect from apoptosis through the IGF-1R receptor while limiting cellular growth through increased expression and activation of TGF-beta1.

Characterization of a new Gsx2-cre line in the developing mouse telencephalon.

PubMed

Qin, Shenyue; Madhavan, Mayur; Waclaw, Ronald R; Nakafuku, Masato; Campbell, Kenneth

2016-10-01

In this study, we generated a transgenic mouse line driving Cre and EGFP expression with two putative cis-regulatory modules (CRMs) (i.e., hs687 and hs678) upstream of the homeobox gene Gsx2 (formerly Gsh2), a critical gene for establishing lateral ganglionic eminence (LGE) identity. The combination of these two CRMs drives transgene expression within the endogenous Gsx2 expression domains along the anterior-posterior neuraxis. By crossing this transgenic line with the Rosa tdTomato (Ai14) reporter mouse line, we observed a unique recombination pattern in the lateral ventral telencephalon, namely the LGE and the dorsal half of the medial GE (MGE), but not in the septum. We found robust recombination in many cell types derived from these embryonic regions, including olfactory bulb and amygdala interneurons and striatal projection neurons from the LGE, as well as cortical interneurons from the MGE and caudal GE (CGE). In summary, this transgenic mouse line represents a new tool for genetic manipulation in the LGE/CGE and the dorsal half of MGE. © 2016 Wiley Periodicals, Inc.

Genistein and bisphenol A exposure cause estrogen receptor 1 to bind thousands of sites in a cell type-specific manner

PubMed Central

Gertz, Jason; Reddy, Timothy E.; Varley, Katherine E.; Garabedian, Michael J.; Myers, Richard M.

2012-01-01

Endogenous estrogens that are synthesized in the body impact gene regulation by activating estrogen receptors in diverse cell types. Exogenous compounds that have estrogenic properties can also be found circulating in the blood in both children and adults. The genome-wide impact of these environmental estrogens on gene regulation is unclear. To obtain an integrated view of gene regulation in response to environmental and endogenous estrogens on a genome-wide scale, we performed ChIP-seq to identify estrogen receptor 1 (ESR1; previously estrogen receptor α) binding sites, and RNA-seq in endometrial cancer cells exposed to bisphenol A (BPA; found in plastics), genistein (GEN; found in soybean), or 17β-estradiol (E2; an endogenous estrogen). GEN and BPA treatment induces thousands of ESR1 binding sites and >50 gene expression changes, representing a subset of E2-induced gene regulation changes. Genes affected by E2 were highly enriched for ribosome-associated proteins; however, GEN and BPA failed to regulate most ribosome-associated proteins and instead enriched for transporters of carboxylic acids. Treatment-dependent changes in gene expression were associated with treatment-dependent ESR1 binding sites, with the exception that many genes up-regulated by E2 harbored a BPA-induced ESR1 binding site but failed to show any expression change after BPA treatment. GEN and BPA exhibited a similar relationship to E2 in the breast cancer line T-47D, where cell type specificity played a much larger role than treatment specificity. Overall, both environmental estrogens clearly regulate gene expression through ESR1 on a genome-wide scale, although with lower potency resulting in less ESR1 binding sites and less gene expression changes compared to the endogenous estrogen, E2. PMID:23019147

Establishment of optimized MDCK cell lines for reliable efflux transport studies.

PubMed

Gartzke, Dominik; Fricker, Gert

2014-04-01

Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Analysis of the subcellular localization of the human histone methyltransferase SETDB1

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

Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp; Gotoh, Eiko; Kawamata, Natsuko

2015-10-02

SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclearmore » export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.« less

A transgenic approach to study argininosuccinate synthetase gene expression

PubMed Central

2014-01-01

Background Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. Results Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage. Thus, the time course of EGFP expression in the transgenic mice resembled that of the human ASS gene. Conclusions We demonstrate that the transgenic mouse system reported here has the merit of sensitivity and direct visualization advantage, and is ideal for annotating temporal and spatial expression profiles and the regulation mode of the ASS gene. PMID:24884799

Isolation of Novel CreERT2-Driver Lines in Zebrafish Using an Unbiased Gene Trap Approach

PubMed Central

Jungke, Peggy; Hammer, Juliane; Hans, Stefan; Brand, Michael

2015-01-01

Gene manipulation using the Cre/loxP-recombinase system has been successfully employed in zebrafish to study gene functions and lineage relationships. Recently, gene trapping approaches have been applied to produce large collections of transgenic fish expressing conditional alleles in various tissues. However, the limited number of available cell- and tissue-specific Cre/CreERT2-driver lines still constrains widespread application in this model organism. To enlarge the pool of existing CreERT2-driver lines, we performed a genome-wide gene trap screen using a Tol2-based mCherry-T2a-CreERT2 (mCT2aC) gene trap vector. This cassette consists of a splice acceptor and a mCherry-tagged variant of CreERT2 which enables simultaneous labeling of the trapping event, as well as CreERT2 expression from the endogenous promoter. Using this strategy, we generated 27 novel functional CreERT2-driver lines expressing in a cell- and tissue-specific manner during development and adulthood. This study summarizes the analysis of the generated CreERT2-driver lines with respect to functionality, expression, integration, as well as associated phenotypes. Our results significantly enlarge the existing pool of CreERT2-driver lines in zebrafish and combined with Cre–dependent effector lines, the new CreERT2-driver lines will be important tools to manipulate the zebrafish genome. PMID:26083735

Establishment of Nephrin Reporter Mice and Use for Chemical Screening

PubMed Central

Tsuchida, Junichi; Matsusaka, Taiji; Ohtsuka, Masato; Miura, Hiromi; Okuno, Yukiko; Asanuma, Katsuhiko; Nakagawa, Takahiko; Yanagita, Motoko

2016-01-01

Nephrin is a critical component of glomerular filtration barrier, which is important to maintain glomerular structure and avoid proteinuria. Downregulation of nephrin expression is commonly observed at early stage of glomerular disorders, suggesting that methods to increase nephrin expression in podocytes may have therapeutic utility. Here, we generated a knockin mouse line carrying single copy of 5.5 kb nephrin promoter controlling expression of enhanced green fluorescent protein (EGFP) at Rosa26 genomic locus (Nephrin-EGFP mouse). In these mice, EGFP was specifically expressed in podocytes. Next, we isolated and cultivated glomeruli from these mice, and developed a protocol to automatically quantitate EGFP expression in cultured glomeruli. EGFP signal was markedly reduced after 5 days of culture but reduction was inhibited by vitamin D treatment. We confirmed that vitamin D increased mRNA and protein expression of endogenous nephrin in cultivated glomeruli. Thus, we generated a mouse line converting nephrin promoter activity into fluorescence, which can be used to screen compounds having activity to enhance nephrin gene expression. PMID:27362433

RXRα and LXR activate two promoters in placenta- and tumor-specific expression of PLAC1

PubMed Central

Chen, Yaohui; Moradin, Adi; Schlessinger, David; Nagaraja, Ramaiah

2011-01-01

PLAC1 expression, first characterized as restricted to developing placenta among normal tissues, is also found in a wide range of tumors and transformed cell lines. To understand the basis for its unusual expression profile, we have analyzed the gene structure and its mode of transcription. We find that the gene has a hitherto unique feature, with two promoters, P1 and P2, separated by 105 kb. P2 has been described before. Here we define P1 and show that it and P2 are activated by RXRα in conjunction with LXRα or LXRβ. In placenta, P2 is the preferred promoter, whereas various tumor cell lines tend to express predominantly either one or the other promoter. Furthermore, when each promoter is fused to a luciferase reporter gene and transfected into cancer cell lines, the promoter corresponding to the more active endogenous promoter is preferentially transcribed. Joint expression of activating nuclear receptors can partially account for the restricted expression of PLAC1 in placenta, and may be co-opted for preferential P1 or P2 PLAC1 expression in various tumor cells. PMID:21937108

GABA stimulates human hepatocellular carcinoma growth through overexpressed GABAA receptor theta subunit

PubMed Central

Li, Yue-Hui; Liu, Yan; Li, Yan-Dong; Liu, Yan-Hong; Li, Feng; Ju, Qiang; Xie, Ping-Li; Li, Guan-Cheng

2012-01-01

AIM: To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC). METHODS: Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang’s liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed. RESULTS: We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ. CONCLUSION: GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC. PMID:22690081

LRRK2 modulates vulnerability to mitochondrial dysfunction in C. elegans

PubMed Central

Saha, Shamol; Guillily, Maria; Ferree, Andrew; Lanceta, Joel; Chan, Diane; Ghosh, Joy; Hsu, Cindy H.; Segal, Lilach; Raghavan, Kesav; Matsumoto, Kunihiro; Hisamoto, Naoki; Kuwahara, Tomoki; Iwatsubo, Takeshi; Moore, Landon; Goldstein, Lee; Cookson, Mark; Wolozin, Benjamin

2009-01-01

Summary Mutations in leucine rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson’s disease. We generated lines of C. elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 expression increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C or kinase dead LRRK2 was less than protection by wild type LRRK2. Knockdown of lrk-1, the endogenous orthologue of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT∷GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the WT line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing WT LRRK2; however loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different than that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition, and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson’s disease. PMID:19625511

Late G1 accumulation after 2 Gy of gamma-irradiation is related to endogenous Raf-1 protein expression and intrinsic radiosensitivity in human cells.

PubMed Central

Warenius, H. M.; Jones, M.; Jones, M. D.; Browning, P. G.; Seabra, L. A.; Thompson, C. C.

1998-01-01

We have previously reported a correlation between high endogenous expression of the protein product of the RAF-1 proto-oncogene, intrinsic cellular radiosensitivity and rapid exit from a G2/M delay induced by 2 Gy of gamma-irradiation. Raf1 is a positive serine/threonine kinase signal transduction factor that relays signals from the cell membrane to the MAP kinase system further downstream and is believed to be involved in an ionizing radiation signal transduction pathway modulating the G1/S checkpoint. We therefore extended our flow cytometric studies to investigate relationships between radiosensitivity, endogenous expression of the Raf1 protein and perturbation of cell cycle checkpoints, leading to alterations in the G1, S and G2/M populations after 2 Gy of gamma-irradiation. Differences in intrinsic radiosensitivity after modulation of the G1/S checkpoint have generally been understood to involve p53 function up to the present time. A role for dominant oncogenes in control of G1/S transit in radiation-treated cells has not been identified previously. Here, we show in 12 human in vitro cancer cell lines that late G1 accumulation after 2 Gy of radiation is related to both Raf1 expression (r = 0.91, P = 0.0001) and the radiosensitivity parameter SF2 (r = -0.71, P = 0.009). PMID:9579826

Germ line transmission of a yeast artificial chromosome spanning the murine [alpha][sub 1](I) collagen locus

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

Strauss, W.M.; Dausman, J.; Beard, C.

Molecular complementation of mutant phenotypes by transgenic technology is a potentially important tool for gene identification. A technology was developed to allow the transfer of a physically intact yeast artificial chromosome (YAC) into the germ line of the mouse. A purified 150-kilobase YAC encompassing the murine gene Col1a1 was efficiently introduced into embryonic stem (ES) cells via lipofection. Chimeric founder mice were derived from two transfected ES cell clones. These chimeras transmitted the full length transgene through the germ line, generating two transgenic mouse strains. Transgene expression was visualized as nascent transcripts in interphase nuclei and quantitated by ribonuclease protectionmore » analysis. Both assays indicated that the transgene was expressed at levels comparable to the endogenous collagen gene. 32 refs., 3 figs., 1 tab.« less

Involvement of plant endogenous ABA in Bacillus megaterium PGPR activity in tomato plants.

PubMed

Porcel, Rosa; Zamarreño, Ángel María; García-Mina, José María; Aroca, Ricardo

2014-01-25

Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil bacteria which benefit plants by improving plant productivity and immunity. The mechanisms involved in these processes include the regulation of plant hormone levels such as ethylene and abscisic acid (ABA). The aim of the present study was to determine whether the activity of Bacillus megaterium PGPR is affected by the endogenous ABA content of the host plant. The ABA-deficient tomato mutants flacca and sitiens and their near-isogenic wild-type parental lines were used. Growth, stomatal conductance, shoot hormone concentration, competition assay for colonization of tomato root tips, and root expression of plant genes expected to be modulated by ABA and PGPR were examined. Contrary to the wild-type plants in which PGPR stimulated growth rates, PGPR caused growth inhibition in ABA-deficient mutant plants. PGPR also triggered an over accumulation of ethylene in ABA-deficient plants which correlated with a higher expression of the pathogenesis-related gene Sl-PR1b. Positive correlation between over-accumulation of ethylene and a higher expression of Sl-PR1b in ABA-deficient mutant plants could indicate that maintenance of normal plant endogenous ABA content may be essential for the growth promoting action of B. megaterium by keeping low levels of ethylene production.

Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells.

PubMed

Manca, Antonella; Sini, Maria Cristina; Izzo, Francesco; Ascierto, Paolo A; Tatangelo, Fabiana; Botti, Gerardo; Gentilcore, Giusy; Capone, Marilena; Mozzillo, Nicola; Rozzo, Carla; Cossu, Antonio; Tanda, Francesco; Palmieri, Giuseppe

2011-06-01

Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells.

PubMed

Legut, Mateusz; Dolton, Garry; Mian, Afsar Ali; Ottmann, Oliver G; Sewell, Andrew K

2018-01-18

Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-β is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αβ and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR-modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer-reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αβ TCR resulted in more efficient redirection of CD4 + and CD8 + T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies. © 2018 by The American Society of Hematology.

Endogenous axon guiding chemorepulsant semaphorin-3F inhibits the growth and metastasis of colorectal carcinoma.

PubMed

Wu, Feng; Zhou, Qi; Yang, Jing; Duan, Guang-jie; Ou, Juan-juan; Zhang, Rong; Pan, Feng; Peng, Qiu-ping; Tan, Hong; Ping, Yi-fang; Cui, You-hong; Qian, Cheng; Yan, Xiao-chu; Bian, Xiu-wu

2011-05-01

To elucidate the role of Semaphorin-3F (SEMA3F), originally described as an axon guiding chemorepulsant implicated in nerve development, in the progression of colorectal carcinoma. SEMA3F and its receptor NRP2 were examined in 72 cases of human colorectal carcinoma specimens and cell lines LoVo, SW480, and SW620 with immunohistochemistry and Western blotting. SEMA3F mRNA expression in the frozen tissue specimens and cell lines was examined with quantitative reverse transcriptase-PCR. Confocal laser scanning microscopy was used for detection of cellular localization of the proteins by immunofluorescent staining. MTT assay, flow cytometry, cell adhesion and migration, and xenografts were used to evaluate biological significance of SEMA3F. SEMA3F was significantly reduced in colorectal carcinoma tissues and cell lines. Overexpression of SEMA3F resulted in reduced proliferation, adhesion to fibronectin, and migratory capability as well as reduced S-phase population and integrin αvβ3 expression of SW480 colon cancer cells. In addition, SEMA3F-overexpressing cells exhibited diminished tumorigenesis when transplanted orthotopically in nude mice and reduced liver metastases. Moreover, transfection of siRNA targeting SEMA3F in colon cancer cells increased their tumorigenicity in vivo. Endogenous SEMA3F acts as a suppressor of the growth and metastasis of human colorectal cancer cells. ©2011 AACR.

Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4.

PubMed

Dale, James; James, Anthony; Paul, Jean-Yves; Khanna, Harjeet; Smith, Mark; Peraza-Echeverria, Santy; Garcia-Bastidas, Fernando; Kema, Gert; Waterhouse, Peter; Mengersen, Kerrie; Harding, Robert

2017-11-14

Banana (Musa spp.) is a staple food for more than 400 million people. Over 40% of world production and virtually all the export trade is based on Cavendish banana. However, Cavendish banana is under threat from a virulent fungus, Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) for which no acceptable resistant replacement has been identified. Here we report the identification of transgenic Cavendish with resistance to TR4. In our 3-year field trial, two lines of transgenic Cavendish, one transformed with RGA2, a gene isolated from a TR4-resistant diploid banana, and the other with a nematode-derived gene, Ced9, remain disease free. Transgene expression in the RGA2 lines is strongly correlated with resistance. Endogenous RGA2 homologs are also present in Cavendish but are expressed tenfold lower than that in our most resistant transgenic line. The expression of these homologs can potentially be elevated through gene editing, to provide non-transgenic resistance.

Gfi1-Cre knock-in mouse line: A tool for inner ear hair cell-specific gene deletion

PubMed Central

Yang, Hua; Gan, Jean; Xie, Xiaoling; Deng, Min; Feng, Liang; Chen, Xiaowei; Gao, Zhiqiang; Gan, Lin

2010-01-01

Summary Gfi1encodes a zinc-finger transcription factor essential for the development and maintenance of haematopoiesis and the inner ear. In mouse inner ear, Gfi1 expression is confined to hair cells during development and in adulthood. To construct a genetic tool for inner ear hair cell-specific gene deletion, we generated a Gfi1-Cre mouse line by knocking-in Cre coding sequences into the Gfi1 locus and inactivating the endogenous Gfi1. The specificity and efficiency of Gfi1-Cre recombinase-mediated recombination in the developing inner ear was revealed through the expression of the conditional R26R-lacZ reporter gene. The onset of lacZ expression in the Gfi1Cre/+ inner ear was first detected at E13.5 in the vestibule and at E15.5 in the cochlea, coinciding with the generation of hair cells. Throughout inner ear development, lacZ expression was detected only in hair cells. Thus, Gfi1-Cre knock-in mouse line provides a useful tool for gene manipulations specifically in inner ear hair cells. PMID:20533399

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

PubMed

Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-02-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells.

Endogenous pyrogen production by Hodgkin's disease and human histiocytic lymphoma cell lines in vitro.

PubMed Central

Bodel, P; Ralph, P; Wenc, K; Long, J C

1980-01-01

Fever not explained by infection may occur in patients with malignant lymphoma presumably caused by a release of endogenous pyrogen. Although pyrogen has been found in some tumors with a mixed cell population, production of endogenous pyrogen by the neoplastic cells has not been demonstrated. This report documents the apparently spontaneous synthesis and release of such pyrogen by two human tumor cell lines derived from patients with Hodgkin's disease and histiocytic lymphoma. The endogenous pyrogen from the two cell lines was similar and closely resembled that produced by normal human monocytes in antigenic properties as well as heat and pronase sensitivity. The Hodgkin's disease and histiocytic lymphoma cell lines do not require specific stimulation for the production of endogenous pyrogen suggesting that the mechanism of pyrogen release by neoplastic macrophage-related cells differs from that of normal phagocytic cells. The tumor-associated fever in some patients with malignant lymphoma may be caused by a release of endogenous pyrogen by proliferating neoplastic cells. PMID:6985918

miRNA-556-3p promotes human bladder cancer proliferation, migration and invasion by negatively regulating DAB2IP expression.

PubMed

Feng, Chen; Sun, Ping; Hu, Jing; Feng, Hua; Li, Mingqiu; Liu, Guibo; Pan, Yanming; Feng, Ying; Xu, Yongliang; Feng, Kejian; Feng, Yukuan

2017-06-01

MicroRNAs (miRNAs) play critical roles in tumorigenesis and metastasis by negatively regulating gene expression through complementary binding to the 3'-untranslated region of target mRNAs. The role of miRNAs in expression of the tumor suppressor DAB2IP in bladder cancer (BC) remains unknown. The aim of the present study was to identify miRNAs targeting DAB2IP and determine their expression and function in BC. We predicted candidate miRNAs targeting DAB2IP using TargetScan software. Dual-luciferase reporter assays confirmed that miRNA-556-3p directly regulated DAB2IP expression. Quantitative RT-PCR and RNase protection assays showed that endogenous miRNA-556-3p expression was significantly upregulated in clinical samples of BC patients and BC cell lines and western blot analysis indicated that DAB2IP expression in BC tissues and BC cell lines was concurrently downregulated. Gain or loss of function studies showed that upregulation of miRNA-556-3p promoted proliferation, invasion, migration and colony formation of BC cells, whereas downregulation resulted in opposite effects. Importantly, restoration of DAB2IP expression rescued the effects induced by miRNA-556-3p. Overexpression of miRNA-556-3p in BC cells not only decreased DAB2IP expression, but also markedly increased Ras GTPase activity and ERK1/2 phosphorylation level. These findings suggest that DAB2IP is a direct target of miRNA-556-3p, and endogenous miRNA-556-3p expression shows inverse correlation with simultaneous DAB2IP expression in BC tissues and cells. miRNA-556-3p functions as a tumor promoter in tumorigenesis and metastasis of BC by targeting DAB2IP. Moreover, miRNA-556-3p-mediated DAB2IP suppression plays an oncogenic role by partial activation of the Ras-ERK pathway.

Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer

PubMed Central

van der Gun, B T F; Huisman, C; Stolzenburg, S; Kazemier, H G; Ruiters, M H J; Blancafort, P; Rots, M G

2013-01-01

Background: The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear. Methods: Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain. Results: Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival. Conclusion: The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer. PMID:23403823

Brain selective transgene expression in zebrafish using an NRSE derived motif

PubMed Central

Bergeron, Sadie A.; Hannan, Markus C.; Codore, Hiba; Fero, Kandice; Li, Grace H.; Moak, Zachary; Yokogawa, Tohei; Burgess, Harold A.

2012-01-01

Transgenic technologies enable the manipulation and observation of circuits controlling behavior by permitting expression of genetically encoded reporter genes in neurons. Frequently though, neuronal expression is accompanied by transgene expression in non-neuronal tissues, which may preclude key experimental manipulations, including assessment of the contribution of neurons to behavior by ablation. To better restrict transgene expression to the nervous system in zebrafish larvae, we have used DNA sequences derived from the neuron-restrictive silencing element (NRSE). We find that one such sequence, REx2, when used in conjunction with several basal promoters, robustly suppresses transgene expression in non-neuronal tissues. Both in transient transgenic experiments and in stable enhancer trap lines, suppression is achieved without compromising expression within the nervous system. Furthermore, in REx2 enhancer trap lines non-neuronal expression can be de-repressed by knocking down expression of the NRSE binding protein RE1-silencing transcription factor (Rest). In one line, we show that the resulting pattern of reporter gene expression coincides with that of the adjacent endogenous gene, hapln3. We demonstrate that three common basal promoters are susceptible to the effects of the REx2 element, suggesting that this method may be useful for confining expression from many other promoters to the nervous system. This technique enables neural specific targeting of reporter genes and thus will facilitate the use of transgenic methods to manipulate circuit function in freely behaving larvae. PMID:23293587

The Receptor Tyrosine Kinase EphA2 Is a Direct Target Gene of Hypermethylated in Cancer 1 (HIC1)*

PubMed Central

Foveau, Bénédicte; Boulay, Gaylor; Pinte, Sébastien; Van Rechem, Capucine; Rood, Brian R.; Leprince, Dominique

2012-01-01

The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically silenced in many human tumors. Here, we show that ectopic expression of HIC1 in the highly malignant MDA-MB-231 breast cancer cell line severely impairs cell proliferation, migration, and invasion in vitro. In parallel, infection of breast cancer cell lines with a retrovirus expressing HIC1 also induces decreased mRNA and protein expression of the tyrosine kinase receptor EphA2. Moreover, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments demonstrate that endogenous HIC1 proteins are bound, together with the MTA1 corepressor, to the EphA2 promoter in WI38 cells. Taken together, our results identify EphA2 as a new direct target gene of HIC1. Finally, we observe that inactivation of endogenous HIC1 through RNA interference in normal breast epithelial cells results in the up-regulation of EphA2 and is correlated with increased cellular migration. To conclude, our results involve the tumor suppressor HIC1 in the transcriptional regulation of the tyrosine kinase receptor EphA2, whose ligand ephrin-A1 is also a HIC1 target gene. Thus, loss of the regulation of this Eph pathway through HIC1 epigenetic silencing could be an important mechanism in the pathogenesis of epithelial cancers. PMID:22184117

Competitive Expression of Endogenous Wheat CENH3 May Lead to Suppression of Alien ZmCENH3 in Transgenic Wheat × Maize Hybrids.

PubMed

Chen, Wei; Zhu, Qilin; Wang, Haiyan; Xiao, Jin; Xing, Liping; Chen, Peidu; Jin, Weiwei; Wang, Xiu-E

2015-11-20

Uniparental chromosome elimination in wheat × maize hybrid embryos is widely used in double haploid production of wheat. Several explanations have been proposed for this phenomenon, one of which is that the lack of cross-species CENH3 incorporation may act as a barrier to interspecies hybridization. However, it is unknown if this mechanism applies universally. To study the role of CENH3 in maize chromosome elimination of wheat × maize hybrid embryos, maize ZmCENH3 and wheat αTaCENH3-B driven by the constitutive CaMV35S promoter were transformed into wheat variety Yangmai 158. Five transgenic lines for ZmCENH3 and six transgenic lines for αTaCENH3-B were identified. RT-PCR analysis showed that the transgene could be transcribed at a low level in all ZmCENH3 transgenic lines, whereas transcription of endogenous wheat CENH3 was significantly up-regulated. Interestingly, the expression levels of both wheat CENH3 and ZmCENH3 in the ZmCENH3 transgenic wheat × maize hybrid embryos were higher than those in the non-transformed Yangmai 158 × maize hybrid embryos. This indicates that the alien ZmCENH3 in wheat may induce competitive expression of endogenous wheat CENH3, leading to suppression of ZmCENH3 over-expression. Eliminations of maize chromosomes in hybrid embryos of ZmCENH3 transgenic wheat × maize and Yangmai 158 × maize were compared by observations on micronuclei presence, by marker analysis using maize SSRs (simple sequence repeats), and by FISH (fluorescence in situ hybridization) using 45S rDNA as a probe. The results indicate that maize chromosome elimination events in the two crosses are not significantly different. Fusion protein ZmCENH3-YFP could not be detected in ZmCENH3 transgenic wheat by either Western blotting or immnunostaining, whereas accumulation and loading of the αTaCENH3-B-GFP fusion protein was normal in αTaCENH3-B transgenic lines. As ZmCENH3-YFP did not accumulate after AM114 treatment, we speculate that low levels of ZmCENH3 protein in transgenic wheat may be one of the factors that lead to failure of suppression of maize chromatin elimination in ZmCENH3 transgenic wheat × maize hybrids. Copyright © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Human snoRNA-93 is processed into a microRNA-like RNA that promotes breast cancer cell invasion.

PubMed

Patterson, Dillon G; Roberts, Justin T; King, Valeria M; Houserova, Dominika; Barnhill, Emmaline C; Crucello, Aline; Polska, Caroline J; Brantley, Lucas W; Kaufman, Garrett C; Nguyen, Michael; Santana, Megann W; Schiller, Ian A; Spicciani, Julius S; Zapata, Anastasia K; Miller, Molly M; Sherman, Timothy D; Ma, Ruixia; Zhao, Hongyou; Arora, Ritu; Coley, Alexander B; Zeidan, Melody M; Tan, Ming; Xi, Yaguang; Borchert, Glen M

2017-01-01

Genetic searches for tumor suppressors have recently linked small nucleolar RNA misregulations with tumorigenesis. In addition to their classically defined functions, several small nucleolar RNAs are now known to be processed into short microRNA-like fragments called small nucleolar RNA-derived RNAs. To determine if any small nucleolar RNA-derived RNAs contribute to breast malignancy, we recently performed a RNA-seq-based comparison of the small nucleolar RNA-derived RNAs of two breast cancer cell lines (MCF-7 and MDA-MB-231) and identified small nucleolar RNA-derived RNAs derived from 13 small nucleolar RNAs overexpressed in MDA-MB-231s. Importantly, we find that inhibiting the most differentially expressed of these small nucleolar RNA-derived RNAs (sdRNA-93) in MDA-MB-231 cells results primarily in a loss of invasiveness, whereas increased sdRNA-93 expression in either cell line conversely results in strikingly enhanced invasion. Excitingly, we recently determined sdRNA-93 expressions in small RNA-seq data corresponding to 116 patient tumors and normal breast controls, and while we find little sdRNA-93 expression in any of the controls and only sporadic expression in most subtypes, we find robust expression of sdRNA-93 in 92.8% of Luminal B Her2+tumors. Of note, our analyses also indicate that at least one of sdRNA-93's endogenous roles is to regulate the expression of Pipox, a sarcosine metabolism-related protein whose expression significantly correlates with distinct molecular subtypes of breast cancer. We find sdRNA-93 can regulate the Pipox 3'UTR via standard reporter assays and that manipulating endogenous sdRNA-93 levels inversely correlates with altered Pipox expression. In summary, our results strongly indicate that sdRNA-93 expression actively contributes to the malignant phenotype of breast cancer through participating in microRNA-like regulation.

A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells.

PubMed

Xue, Haipeng; Wu, Sen; Papadeas, Sophia T; Spusta, Steve; Swistowska, Anna Maria; MacArthur, Chad C; Mattson, Mark P; Maragakis, Nicholas J; Capecchi, Mario R; Rao, Mahendra S; Zeng, Xianmin; Liu, Ying

2009-08-01

In this study, we targeted Olig2, a basic helix-loop-helix transcription factor that plays an important role in motoneuron and oligodendrocyte development, in human embryonic stem cell (hESC) line BG01 by homologous recombination. One allele of Olig2 locus was replaced by a green fluorescent protein (GFP) cassette with a targeting efficiency of 5.7%. Targeted clone R-Olig2 (like the other clones) retained pluripotency, typical hESC morphology, and a normal parental karyotype 46,XY. Most importantly, GFP expression recapitulated endogenous Olig2 expression when R-Olig2 was induced by sonic hedgehog and retinoic acid, and GFP-positive cells could be purified by fluorescence-activated cell sorting. Consistent with previous reports on rodents, early GFP-expressing cells appeared biased to a neuronal fate, whereas late GFP-expressing cells appeared biased to an oligodendrocytic fate. This was corroborated by myoblast coculture, transplantation into the rat spinal cords, and whole genome expression profiling. The present work reports an hESC reporter line generated by homologous recombination targeting a neural lineage-specific gene, which can be differentiated and sorted to obtain pure neural progenitor populations.

Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3

PubMed Central

Zhou, Gaofeng; Ryan, Peter R.

2014-01-01

Malate and citrate efflux from root apices is a mechanism of Al3+ tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley (Hordeum vulgare) is among the most Al3+-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al3+ tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al3+-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al3+-activated citrate efflux from root apices and greater tolerance to Al3+ toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al3+ tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al3+ tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al3+ tolerance of an important crop species. PMID:24692647

Impact of the underlying mutation and the route of vector administration on immune responses to factor IX in gene therapy for hemophilia B.

PubMed

Cao, Ou; Hoffman, Brad E; Moghimi, Babak; Nayak, Sushrusha; Cooper, Mario; Zhou, Shangzhen; Ertl, Hildegund C J; High, Katherine A; Herzog, Roland W

2009-10-01

Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.

Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.

PubMed

Gartzke, Dominik; Delzer, Jürgen; Laplanche, Loic; Uchida, Yasuo; Hoshi, Yutaro; Tachikawa, Masanori; Terasaki, Tetsuya; Sydor, Jens; Fricker, Gert

2015-06-01

To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks. Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones. From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate. Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis TOM1.

PubMed

Ali, Md Emran; Ishii, Yuko; Taniguchi, Jyun-Ichi; Waliullah, Sumyya; Kobayashi, Kappei; Yaeno, Takashi; Yamaoka, Naoto; Nishiguchi, Masamichi

2018-05-01

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.

Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.

PubMed

Kaminuma, Osamu; Katayama, Kazufumi; Inoue, Kimiko; Saeki, Mayumi; Nishimura, Tomoe; Kitamura, Noriko; Shimo, Yusuke; Tofukuji, Soichi; Ishida, Satoru; Ogonuki, Narumi; Kamimura, Satoshi; Oikawa, Mami; Katoh, Shigeki; Mori, Akio; Shichijo, Michitaka; Hiroi, Takachika; Ogura, Atsuo

2017-06-01

T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4 + T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4 + T-cell-mediated pathogenesis and cellular commitment in immune diseases. © 2017 The Authors.

The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells

NASA Astrophysics Data System (ADS)

Anderson, Alison M.; Kalimutho, Murugan; Harten, Sarah; Nanayakkara, Devathri M.; Khanna, Kum Kum; Ragan, Mark A.

2017-01-01

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

Expression and Functional Significance of HtrA1 Loss in Endometrial Cancer

PubMed Central

Mullany, Sally A.; Moslemi-Kebria, Mehdi; Rattan, Ramandeep; Khurana, Ashwani; Clayton, Amy; Ota, Takayo; Mariani, Andrea; Podratz, Karl C.; Chien, Jeremy; Shridhar, Viji

2010-01-01

Purpose The purpose of this study was to determine if loss of serine protease HtrA1 in endometrial cancer will promote the invasive potential of EC cell lines. Experimental design Western blot analysis and immunohistochemistry methods were used to determine HtrA1 expression in EC cell lines and primary tumors, respectively. Migration, invasion assays and in vivo xenograft experiment were performed to compare the extent of metastasis between HtrA1 expressing and HtrA-1 knocked down clones. Results Western blot analysis of HtrA1 in 13 EC cell lines revealed complete loss of HtrA1 expression in all 7 papillary serous EC cell lines. Downregulation of HtrA1 in Hec1A and Hec1B cell lines resulted in a 3-4 fold increase in the invasive potential. Exogenous expression of HtrA1 in Ark 1 and Ark 2 cells resulted in 3-4 fold decrease in both invasive and migration potential of these cells. There was an increased rate of metastasis to the lungs associated with HtrA1 downregulation in Hec1B cells compared to control cells with endogenous HtrA1 expression. Enhanced expression of HtrA1 in Ark 2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or protease deficient (SA mutant) Ark 2 cells. Immunohistochemical (IHC) analysis showed 57% (105/184) of primary EC tumors had low HtrA1 expression. The association of low HtrA1 expression with high-grade endometrioid tumors was statistically significant (p=0.016). Conclusions Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors. PMID:21098697

Differential hormonal and gene expression dynamics in two inbred sunflower lines with contrasting dormancy level.

PubMed

Roselló, Paula L; Vigliocco, Ana E; Andrade, Andrea M; Riera, Natalí V; Calafat, Mario; Molas, María L; Alemano, Sergio G

2016-05-01

Seed germination and dormancy are tightly regulated by hormone metabolism and signaling pathway. We investigated the endogenous content of abscisic acid (ABA), its catabolites, and gibberellins (GAs), as well as the expression level of certain ABA and GAs metabolic and signaling genes in embryo of dry and imbibed cypselas of inbred sunflower (Helianthus annuus L., Asteraceae) lines: B123 (dormant) and B91 (non-dormant). Under our experimental conditions, the expression of RGL2 gene might be related to the ABA peak in B123 line at 3 h of imbibition. Indeed, RGL2 transcripts are absent in dry and early embedded cypselas of the non-dormant line B91. ABA increase was accompanied by a significant ABA-Glucosyl ester (ABA-GE) and phaseic acid (PA) (two ABA catabolites) decrease in B123 line (3 h) which indicates that ABA metabolism seems to be more active in this line, and that it would be involved in the imposition and maintenance of sunflower seed dormancy, as it has been reported for many species. Finally, an increase of bioactive GAs (GA1 and GA3) occurs at 12 h of imbibition in both lines after a decrease in ABA content. This study shows the first report about the RGL2 tissue-specific gene expression in sunflower inbred lines with contrasting dormancy level. Furthermore, our results provide evidence that ABA and GAs content and differential expression of metabolism and signaling genes would be interacting in seed dormancy regulation through a mechanism of action related to embryo itself. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

An immortal cell line to study the role of endogenous CFTR in electrolyte absorption.

PubMed

Bell, C L; Quinton, P M

1995-01-01

The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of "endogenous" CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed two fundamental components of absorption, a cAMP activated Cl- conductance (GCl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10(-5) M) significantly increased the Cl- diffusion potential (Vt) generated by a luminally directed Cl- gradient from -15.3 +/- 0.7 mV to -23.9 +/- 1.1 mV, n = 39; and decreased the transepithelial resistance (Rt) from 814.8 +/- 56.3 omega.cm2 to 750.5 +/- 47.5 omega.cm2, n = 39, (n = number of cultures). cAMP activation, anion selectivity (Cl- > I- > gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10(-5) M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 +/- 3.1% and 77.9 +/- 2.6%, respectively, and an increase in Rt by 7.2 +/- 0.8%, n = 36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin induces alpha1B-adrenergic receptor phosphorylation and desensitization.

PubMed

García-Sáinz, J Adolfo; Romero-Avila, M Teresa; Molina-Muñoz, Tzindilú; Medina, Luz del Carmen

2004-09-03

The ability of insulin to induce alpha1B-adrenoceptor phosphorylation and desensitization was tested in two model systems: rat-1 cells that stably express alpha1B-adrenoceptors, through transfection, and endogenously express insulin receptors and DDT1 MF2 cells that endogenously express both receptors. Insulin induced concentration-dependent increases in the phosphorylation state of the adrenergic receptors in the two models with similar EC50 values (0.5-2 nM). The effect was rapid in the two systems but it was sustained in rat-1 cells and transient in DDT1 MF2 cells. In both cell lines, the insulin-mediated phosphorylation of alpha1B-adrenoceptors was blocked by wortmannin and LY 294002, and by staurosporine and bisindolylmaleimide I, indicating that the effect involved phosphoinositide 3-kinase and protein kinase C activities. The adrenoceptor phosphorylation induced by insulin was associated to desensitization as evidences by a diminished elevation of intracellular calcium in response to noradrenaline. Inhibitors of phosphoinositide 3-kinase and protein kinase C blocked the functional desensitization induced by insulin.

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

PubMed

Patel, Utsav A; Patel, Amrutlal K; Joshi, Chaitanya G

2015-01-01

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers.

Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

PubMed

Bian, Yue-Hong; Xu, Cheng; Li, Junling; Xu, Jin; Zhang, Hongwei; Du, Shao Jun

2011-08-01

Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

Expression of a Drosophila glutathione transferase in Arabidopsis confers the ability to detoxify the environmental pollutant, and explosive, 2,4,6-trinitrotoluene.

PubMed

Tzafestas, Kyriakos; Razalan, Maria M; Gyulev, Ivan; Mazari, Aslam M A; Mannervik, Bengt; Rylott, Elizabeth L; Bruce, Neil C

2017-04-01

The explosive 2,4,6-trinitrotoluene (TNT) is a significant, global environmental pollutant that is both toxic and recalcitrant to degradation. Given the sheer scale and inaccessible nature of contaminated areas, phytoremediation may be a viable clean-up approach. Here, we have characterized a Drosophila melanogaster glutathione transferase (DmGSTE6) which has activity towards TNT. Recombinantly expressed, purified DmGSTE6 produces predominantly 2-glutathionyl-4,6-dinitrotoluene, and has a 2.5-fold higher Maximal Velocity (V max ), and five-fold lower Michaelis Constant (K m ) than previously characterized TNT-active Arabidopsis thaliana (Arabidopsis) GSTs. Expression of DmGSTE6 in Arabidopsis conferred enhanced resistance to TNT, and increased the ability to remove TNT from contaminated soil relative to wild-type plants. Arabidopsis lines overexpressing TNT-active GSTs AtGST-U24 and AtGST-U25 were compromised in biomass production when grown in the absence of TNT. This yield drag was not observed in the DmGSTE6-expressing Arabidopsis lines. We hypothesize that increased levels of endogenous TNT-active GSTs catalyse excessive glutathionylation of endogenous substrates, depleting glutathione pools, an activity that DmGST may lack. In conclusion, DmGSTE6 has activity towards TNT, producing a compound with potential for further biodegradation. Selecting or manipulating plants to confer DmGSTE6-like activity could contribute towards development of phytoremediation strategies to clean up TNT from polluted military sites. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

BAC Recombineering of the Agouti Loci from Spotted Gar and Zebrafish Reveals the Evolutionary Ancestry of Dorsal-Ventral Pigment Asymmetry in Fish.

PubMed

Cal, Laura; MegÍas, Manuel; Cerdá-Reverter, José Miguel; Postlethwait, John H; Braasch, Ingo; Rotllant, Josep

2017-11-01

Dorsoventral pigment patterning, characterized by a light ventrum and a dark dorsum, is one of the most widespread chromatic adaptations in vertebrate body coloration. In mammals, this countershading depends on differential expression of agouti-signaling protein (ASIP), which drives a switch of synthesis of one type of melanin to another within melanocytes. Teleost fish share countershading, but the pattern results from a differential distribution of multiple types of chromatophores, with black-brown melanophores most abundant in the dorsal body and reflective iridophores most abundant in the ventral body. We previously showed that Asip1 (a fish ortholog of mammalian ASIP) plays a role in patterning melanophores. This observation leads to the surprising hypothesis that agouti may control an evolutionarily conserved pigment pattern by regulating different mechanisms in mammals and fish. To test this hypothesis, we compared two ray-finned fishes: the teleost zebrafish and the nonteleost spotted gar (Lepisosteus oculatus). By examining the endogenous pattern of asip1 expression in gar, we demonstrate a dorsoventral-graded distribution of asip1 expression that is highest ventrally, similar to teleosts. Additionally, in the first reported experiments to generate zebrafish transgenic lines carrying a bacterial artificial chromosome (BAC) from spotted gar, we show that both transgenic zebrafish lines embryos replicate the endogenous asip1 expression pattern in adult zebrafish, showing that BAC transgenes from both species contain all of the regulatory elements required for regular asip1 expression within adult ray-finned fishes. These experiments provide evidence that the mechanism leading to an environmentally important pigment pattern was likely in place before the origin of teleosts. © 2017 Wiley Periodicals, Inc.

Identification and characterization of TF1(phox), a DNA-binding protein that increases expression of gp91(phox) in PLB985 myeloid leukemia cells.

PubMed

Eklund, E A; Kakar, R

1997-04-04

The CYBB gene encodes gp91(phox), the heavy chain of the phagocyte-specific NADPH oxidase. CYBB is transcriptionally inactive until the promyelocyte stage of myelopoiesis, and in mature phagocytes, expression of gp91(phox) is further increased by interferon-gamma (IFN-gamma) and other inflammatory mediators. The CYBB promoter region contains several lineage-specific cis-elements involved in the IFN-gamma response. We screened a leukocyte cDNA expression library for proteins able to bind to one of these cis-elements (-214 to -262 base pairs) and identified TF1(phox), a protein with sequence-specific binding to the CYBB promoter. Electrophoretic mobility shift assay with nuclear proteins from a variety of cell lines demonstrated binding of a protein to the CYBB promoter that was cross-immunoreactive with TF1(phox). DNA binding of this protein was increased by IFN-gamma treatment in the myeloid cell line PLB985, but not in the non-myeloid cell line HeLa. Overexpression of recombinant TF1(phox) in PLB985 cells increased endogenous gp91(phox) message abundance, but did not lead to cellular differentiation. Overexpression of TF1(phox) in myeloid leukemia cell lines increased reporter gene expression from artificial promoter constructs containing CYBB promoter sequence. These data suggested that TF1(phox) increased expression of gp91(phox).

Entrainment of Breast Cell Lines Results in Rhythmic Fluctuations of MicroRNAs

PubMed Central

Chacolla-Huaringa, Rafael; Trevino, Victor; Scott, Sean-Patrick

2017-01-01

Circadian rhythms are essential for temporal (~24 h) regulation of molecular processes in diverse species. Dysregulation of circadian gene expression has been implicated in the pathogenesis of various disorders, including hypertension, diabetes, depression, and cancer. Recently, microRNAs (miRNAs) have been identified as critical modulators of gene expression post-transcriptionally, and perhaps involved in circadian clock architecture or their output functions. The aim of the present study is to explore the temporal expression of miRNAs among entrained breast cell lines. For this purpose, we evaluated the temporal (28 h) expression of 2006 miRNAs in MCF-10A, MCF-7, and MDA-MB-231 cells using microarrays after serum shock entrainment. We noted hundreds of miRNAs that exhibit rhythmic fluctuations in each breast cell line, and some of them across two or three cell lines. Afterwards, we validated the rhythmic profiles exhibited by miR-141-5p, miR-1225-5p, miR-17-5p, miR-222-5p, miR-769-3p, and miR-548ay-3p in the above cell lines, as well as in ZR-7530 and HCC-1954 using RT-qPCR. Our results show that serum shock entrainment in breast cells lines induces rhythmic fluctuations of distinct sets of miRNAs, which have the potential to be related to endogenous circadian clock, but extensive investigation is required to elucidate that connection. PMID:28704935

Establishment of a Novel Permissive Cell Line for the Propagation of Hepatitis C Virus by Expression of MicroRNA miR122

PubMed Central

Kambara, Hiroto; Fukuhara, Takasuke; Shiokawa, Mai; Ono, Chikako; Ohara, Yuri; Kamitani, Wataru

2012-01-01

The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of “cured” cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics. PMID:22114337

Enhancing the aluminium tolerance of barley by expressing the citrate transporter genes SbMATE and FRD3.

PubMed

Zhou, Gaofeng; Pereira, Jorge F; Delhaize, Emmanuel; Zhou, Meixue; Magalhaes, Jurandir V; Ryan, Peter R

2014-06-01

Malate and citrate efflux from root apices is a mechanism of Al(3+) tolerance in many plant species. Citrate efflux is facilitated by members of the MATE (multidrug and toxic compound exudation) family localized to the plasma membrane of root cells. Barley (Hordeum vulgare) is among the most Al(3+)-sensitive cereal species but the small genotypic variation in tolerance that is present is correlated with citrate efflux via a MATE transporter named HvAACT1. This study used a biotechnological approach to increase the Al(3+) tolerance of barley by transforming it with two MATE genes that encode citrate transporters: SbMATE is the major Al(3+)-tolerance gene from sorghum whereas FRD3 is involved with Fe nutrition in Arabidopsis. Independent transgenic and null T3 lines were generated for both transgenes. Lines expressing SbMATE showed Al(3+)-activated citrate efflux from root apices and greater tolerance to Al(3+) toxicity than nulls in hydroponic and short-term soil trials. Transgenic lines expressing FRD3 exhibited similar phenotypes except citrate release from roots occurred constitutively. The Al(3+) tolerance of these lines was compared with previously generated transgenic barley lines overexpressing the endogenous HvAACT1 gene and the TaALMT1 gene from wheat. Barley lines expressing TaALMT1 showed significantly greater Al(3+) tolerance than all lines expressing MATE genes. This study highlights the relative efficacy of different organic anion transport proteins for increasing the Al(3+) tolerance of an important crop species. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

OsRACK1 Is Involved in Abscisic Acid- and H2O2-Mediated Signaling to Regulate Seed Germination in Rice (Oryza sativa, L.)

PubMed Central

Zhang, Dongping; Chen, Li; Li, Dahong; Lv, Bing; Chen, Yun; Chen, Jingui; XuejiaoYan; Liang, Jiansheng

2014-01-01

The receptor for activated C kinase 1 (RACK1) is one member of the most important WD repeat–containing family of proteins found in all eukaryotes and is involved in multiple signaling pathways. However, compared with the progress in the area of mammalian RACK1, our understanding of the functions and molecular mechanisms of RACK1 in the regulation of plant growth and development is still in its infancy. In the present study, we investigated the roles of rice RACK1A gene (OsRACK1A) in controlling seed germination and its molecular mechanisms by generating a series of transgenic rice lines, of which OsRACK1A was either over-expressed or under-expressed. Our results showed that OsRACK1A positively regulated seed germination and negatively regulated the responses of seed germination to both exogenous ABA and H2O2. Inhibition of ABA biosynthesis had no enhancing effect on germination, whereas inhibition of ABA catabolism significantly suppressed germination. ABA inhibition on seed germination was almost fully recovered by exogenous H2O2 treatment. Quantitative analyses showed that endogenous ABA levels were significantly higher and H2O2 levels significantly lower in OsRACK1A-down regulated transgenic lines as compared with those in wildtype or OsRACK1A-up regulated lines. Quantitative real-time PCR analyses showed that the transcript levels of OsRbohs and amylase genes, RAmy1A and RAmy3D, were significantly lower in OsRACK1A-down regulated transgenic lines. It is concluded that OsRACK1A positively regulates seed germination by controlling endogenous levels of ABA and H2O2 and their interaction. PMID:24865690

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

PubMed Central

Achour, Lamia; Scott, Mark G.H.; Shirvani, Hamasseh; Thuret, Alain; Bismuth, Georges; Labbé-Jullié, Catherine; Marullo, Stefano

2009-01-01

The association of CD4, a glycoprotein involved in T cell development and antigen recognition, and CCR5, a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for the human immunodeficiency virus HIV. We observed that the vast majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relative low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration–dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cell distribution of CXCR4, the other HIV co-receptor. These results reveal a previously unappreciated role of CD4, which contributes to regulate CCR5 export to the plasma membrane. PMID:19064722

MicroRNA-218 inhibits the proliferation of human choriocarcinoma JEG-3 cell line by targeting Fbxw8

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

Shi, Dazun; Tan, Zhihui; Lu, Rong

2014-08-08

Highlights: • The miR-218 expression was decreased in choriocarcinoma cell lines. • The Fbxw8 protein expression was increased in choriocarcinoma cell lines. • We show that Fbxw8 is bona-fide target of miR-218 in JEG-3. • Ectopic miR-218 expression inhibits the proliferation of JEG-3 via Fbxw8. • Overexpression of miR-218 affected cyclin A and p27 expression via Fbxw8. - Abstract: MicroRNAs (miRNAs) are endogenous 19–25 nucleotide noncoding single-stranded RNAs that regulate gene expression by blocking the translation or decreasing the stability of mRNAs. In this study, we showed that miR-218 expression levels were decreased while Fbxw8 expression levels were increased inmore » human choriocarcinoma cell lines, and identified Fbxw8 as a novel direct target of miR-218. Overexpression of miR-218 inhibited cell growth arrest at G2/M phase, suppressed the protein levels of cyclin A and up-regulated the expression levels of p27 through decreasing the levels of Fbxw8. On the other hand, forced expression of Fbxw8 partly rescued the effect of miR-218 in the cells, attenuated cell proliferation decrease the percentage of cells at G2/M phase, induced cyclin A protein expression and suppressed the protein level of p27 through up-regulating the levels of Fbxw8. Taken together, these findings will shed light the role to mechanism of miR-218 in regulating JEG-3 cells proliferation via miR-218/Fbxw8 axis, and miR-218 may serve as a novel potential therapeutic target in human choriocarcinoma in the future.« less

Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

PubMed

Das, Krishna; Eisel, David; Lenkl, Clarissa; Goyal, Ashish; Diederichs, Sven; Dickes, Elke; Osen, Wolfram; Eichmüller, Stefan B

2017-01-01

In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1.

PubMed

Deshet, Naamit; Lupu-Meiri, Monica; Espinoza, Ingrid; Fili, Oded; Shapira, Yuval; Lupu, Ruth; Gershengorn, Marvin C; Oron, Yoram

2008-09-01

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.

Direct modulation of TRPM4 and TRPM3 channels by the phospholipase C inhibitor U73122

PubMed Central

Michel, Niklas; Behrendt, Marc; Dierich, Marlen; Dembla, Sandeep; Wilke, Bettina U; Konrad, Maik; Lindner, Moritz; Oberwinkler, Johannes; Oliver, Dominik

2016-01-01

Background and Purpose Signalling through phospholipase C (PLC) controls many cellular processes. Much information on the relevance of this important pathway has been derived from pharmacological inhibition of the enzymatic activity of PLC. We found that the most frequently employed PLC inhibitor, U73122, activates endogenous ionic currents in widely used cell lines. Given the extensive use of U73122 in research, we set out to identify these U73122‐sensitive ion channels. Experimental Approach We performed detailed biophysical analysis of the U73122‐induced currents in frequently used cell lines. Key Results At concentrations required to inhibit PLC, U73122 modulated the activity of transient receptor potential melastatin (TRPM) channels through covalent modification. U73122 was shown to be a potent agonist of ubiquitously expressed TRPM4 channels and activated endogenous TRPM4 channels in CHO cells independently of PLC and of the downstream second messengers PI(4,5)P2 and Ca2+. U73122 also potentiated Ca2 +‐dependent TRPM4 currents in human Jurkat T‐cells, endogenous TRPM4 in HEK293T cells and recombinant human TRPM4. In contrast to TRPM4, TRPM3 channels were inhibited whereas the closely related TRPM5 channels were insensitive to U73122, showing that U73122 exhibits high specificity within the TRPM channel family. Conclusions and Implications Given the widespread expression of TRPM4 and TRPM3 channels, these actions of U73122 must be considered when interpreting its effects on cell function. U73122 may also be useful for identifying and characterizing TRPM channels in native tissue, thus facilitating the analysis of their physiology. PMID:27328745

Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

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

Thompson, Pamela D.; Sakwe, Amos; Koumangoye, Rainelli

2014-02-15

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels ofmore » AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head and neck squamous cell carcinoma cell lines synthesize and secret AHSG. • AHSG depleted cell lines are significantly inhibited in their ability to proliferate, adhere, migrate, invade and protect MMP-9. • Human AHSG and bovine fetuin-A are functionally equivalent in regards to growth promotion of cancer cell lines.« less

Modulation of Glucose Transporter 1 (GLUT1) Expression Levels Alters Mouse Mammary Tumor Cell Growth In Vitro and In Vivo

PubMed Central

Young, Christian D.; Lewis, Andrew S.; Rudolph, Michael C.; Ruehle, Marisa D.; Jackman, Matthew R.; Yun, Ui J.; Ilkun, Olesya; Pereira, Renata; Abel, E. Dale; Anderson, Steven M.

2011-01-01

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential. Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1. These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo. PMID:21826239

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

PubMed Central

Flores, Omar; Kennedy, Edward M.; Skalsky, Rebecca L.; Cullen, Bryan R.

2014-01-01

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function. PMID:24464996

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential.

PubMed

Flores, Omar; Kennedy, Edward M; Skalsky, Rebecca L; Cullen, Bryan R

2014-04-01

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

c-kit+ Cells Minimally Contribute Cardiomyocytes to the Heart

PubMed Central

van Berlo, Jop H.; Kanisicak, Onur; Maillet, Marjorie; Vagnozzi, Ronald J.; Karch, Jason; Lin, Suh-Chin J.; Middleton, Ryan C.; Marbán, Eduardo; Molkentin, Jeffery D.

2014-01-01

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine if endogenous c-kit+ cells contribute differentiated cardiomyocytes to the heart during development, with aging or after injury in adulthood. A cDNA encoding either Cre recombinase or a tamoxifen inducible MerCreMer chimeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit+ cells did produce new cardiomyocytes within the heart, although at a percentage of ≈0.03% or less, and if a preponderance towards cellular fusion is considered, the percentage falls below ≈0.008%. In contrast, c-kit+ cells amply generated cardiac endothelial cells. Thus, endogenous c-kit+ cells can generate cardiomyocytes within the heart, although likely at a functionally insignificant level. PMID:24805242

PAX3 is expressed in the stromal compartment of the developing kidney and in Wilms tumors with myogenic phenotype.

PubMed

Hueber, Pierre-Alain; Fukuzawa, Ryuji; Elkares, Reyhan; Chu, Leelee; Blumentkrantz, Miriam; He, Shu-Jie; Anaka, Matthew R; Reeve, Anthony E; Eccles, Michael; Jabado, Nada; Iglesias, Diana M; Goodyer, Paul R

2009-01-01

Wilms tumor (WT) is the most frequent renal neoplasm of childhood; a myogenic component is observed in 5% to 10% of tumors. We demonstrate for the first time that myogenic WTs are associated with expression of PAX3, a transcription factor known to specify myoblast cell fate during muscle development. In a panel of 20 WTs, PAX3 was identified in 13 of 13 tumor samples with myogenic histopathology but was absent in 7 of 7 tumors lacking a myogenic component. Furthermore, we show that PAX3 is expressed in the metanephric mesenchyme and stromal compartment of developing mouse kidney. Modulation of endogenous PAX3 expression in human embryonic kidney (HEK293) cells influenced cell migration in in vitro assays. Mutations of WT1 were consistently associated with PAX3 expression in WTs, and modulation of WT1 expression in HEK293 cells was inversely correlated with the level of endogenous PAX3 protein. We demonstrate abundant PAX3 and absence of PAX2 expression in a novel cell line (WitP3) isolated from the stromal portion of a WT bearing a homozygous deletion of the WT1 gene. We hypothesize that PAX3 sets stromal cell fate in developing kidney but is normally suppressed by WT1 during the mesenchyme-to-epithelium transition leading to nephrogenesis. Loss of WT1 permits aberrant PAX3 expression in a subset of WTs with myogenic phenotype.

Up-regulating the abscisic acid inactivation gene ZmABA8ox1b contributes to seed germination heterosis by promoting cell expansion.

PubMed

Li, Yangyang; Wang, Cheng; Liu, Xinye; Song, Jian; Li, Hongjian; Sui, Zhipeng; Zhang, Ming; Fang, Shuang; Chu, Jinfang; Xin, Mingming; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

2016-04-01

Heterosis has been widely used in agriculture, but the underlying molecular principles are still largely unknown. During seed germination, we observed that maize (Zea mays) hybrid B73/Mo17 was less sensitive than its parental inbred lines to exogenous abscisic acid (ABA), and endogenous ABA content in hybrid embryos decreased more rapidly than in the parental inbred lines. ZmABA8ox1b, an ABA inactivation gene, was consistently more highly up-regulated in hybrid B73/Mo17 than in its parental inbred lines at early stages of seed germination. Moreover, ectopic expression of ZmABA8ox1b obviously promoted seed germination in Arabidopsis Remarkably, microscopic observation revealed that cell expansion played a major role in the ABA-mediated maize seed germination heterosis, which could be attributed to the altered expression of cell wall-related genes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Expression of the cancer-testis antigen BORIS correlates with prostate cancer.

PubMed

Cheema, Zubair; Hari-Gupta, Yukti; Kita, Georgia-Xanthi; Farrar, Dawn; Seddon, Ian; Corr, John; Klenova, Elena

2014-02-01

BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen (CT) family. BORIS is normally present at high levels in the testis; however it is aberrantly expressed in various tumors and cancer cell lines. The main objectives of this study were to investigate BORIS expression together with sub-cellular localization in both prostate cell lines and tumor tissues, and assess correlations between BORIS and clinical/pathological characteristics. We examined BORIS mRNA expression, protein levels and cellular localization in a panel of human prostate tissues, cancer and benign, together with a panel prostate cell lines. We also compared BORIS levels and localization with clinical/pathological characteristics in prostate tumors. BORIS was detected in all inspected prostate cancer cell lines and tumors, but was absent in benign prostatic hyperplasia. Increased levels of BORIS protein positively correlated with Gleason score, T-stage and androgen receptor (AR) protein levels in prostate tumors. The relationship between BORIS and AR was further highlighted in prostate cell lines by the ability of ectopically expressed BORIS to activate the endogenous AR mRNA and protein. BORIS localization in the nucleus plus cytoplasm was also associated with higher BORIS levels and Gleason score. Detection of BORIS in prostate tumors suggests potential applications of BORIS as a biomarker for prostate cancer diagnosis, as an immunotherapy target and, potentially, a prognostic marker of more aggressive prostate cancer. The ability of BORIS to activate the AR gene indicates BORIS involvement in the growth and development of prostate tumors. © 2013 Wiley Periodicals, Inc.

Networking Senescence-Regulating Pathways by Using Arabidopsis Enhancer Trap Lines1

PubMed Central

He, Yuehui; Tang, Weining; Swain, Johnnie D.; Green, Anthony L.; Jack, Thomas P.; Gan, Susheng

2001-01-01

The last phase of leaf development, generally referred to as leaf senescence, is an integral part of plant development that involves massive programmed cell death. Due to a sharp decline of photosynthetic capacity in a leaf, senescence limits crop yield and forest plant biomass production. However, the biochemical components and regulatory mechanisms underlying leaf senescence are poorly characterized. Although several approaches such as differential cDNA screening, differential display, and cDNA subtraction have been employed to isolate senescence-associated genes (SAGs), only a limited number of SAGs have been identified, and information regarding the regulation of these genes is fragmentary. Here we report on the utilization of enhancer trap approach toward the identification and analysis of SAGs. We have developed a sensitive large-scale screening method and have screened 1,300 Arabidopsis enhancer trap lines and have identified 147 lines in which the reporter gene GUS (β-glucuronidase) is expressed in senescing leaves but not in non-senescing ones. We have systematically analyzed the regulation of β-glucuronidase expression in 125 lines (genetically, each contains single T-DNA insertion) by six senescence-promoting factors, namely abscisic acid, ethylene, jasmonic acid, brassinosteroid, darkness, and dehydration. This analysis not only reveals the complexity of the regulatory circuitry but also allows us to postulate the existence of a network of senescence-promoting pathways. We have also cloned three SAGs from randomly selected enhancer trap lines, demonstrating that reporter expression pattern reflects the expression pattern of the endogenous gene. PMID:11402199

Regulation of GluR2 promoter activity by neurotrophic factors via a neuron-restrictive silencer element.

PubMed

Brené, S; Messer, C; Okado, H; Hartley, M; Heinemann, S F; Nestler, E J

2000-05-01

The AMPA glutamate receptor subunit GluR2, which plays a critical role in regulation of AMPA channel function, shows altered levels of expression in vivo after several chronic perturbations. To evaluate the possibility that transcriptional mechanisms are involved, we studied a 1254-nucleotide fragment of the 5'-promoter region of the mouse GluR2 gene in neural-derived cell lines. We focused on regulation of GluR2 promoter activity by two neurotrophic factors, which are known to be altered in vivo in some of the same systems that show GluR2 regulation. Glial-cell line derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) both induced GluR2 promoter activity. This was associated with increased expression of endogenous GluR2 immunoreactivity in the cells as measured by Western blotting. The effect of GDNF and BDNF appeared to be mediated via a NRSE (neuron-restrictive silencer element) present within the GluR2 promoter. The response to these neurotrophic factors was lost upon mutating or deleting this site, but not several other putative response elements present within the promoter. Moreover, overexpression of REST (restrictive element silencer transcription factor; also referred to as NRSF or neuron restrictive silencer factor), which is known to act on NRSEs in other genes to repress gene expression, blocked the ability of GDNF to induce GluR2 promoter activity. However, GDNF did not alter endogenous levels of REST in the cells. Together, these findings suggest that GluR2 expression can be regulated by neurotrophic factors via an apparently novel mechanism involving the NRSE present within the GluR2 gene promoter.

Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

PubMed

Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

2015-01-01

The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in multipotent zebrafish cell cultures and for iPS cell preparation.

Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

PubMed

Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

2017-06-01

We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

Cytokinins influence root gravitropism via differential regulation of auxin transporter expression and localization in Arabidopsis.

PubMed

Pernisova, Marketa; Prat, Tomas; Grones, Peter; Harustiakova, Danka; Matonohova, Martina; Spichal, Lukas; Nodzynski, Tomasz; Friml, Jiri; Hejatko, Jan

2016-10-01

Redirection of intercellular auxin fluxes via relocalization of the PIN-FORMED 3 (PIN3) and PIN7 auxin efflux carriers has been suggested to be necessary for the root gravitropic response. Cytokinins have also been proposed to play a role in controlling root gravitropism, but conclusive evidence is lacking. We present a detailed study of the dynamics of root bending early after gravistimulation, which revealed a delayed gravitropic response in transgenic lines with depleted endogenous cytokinins (Pro35S:AtCKX) and cytokinin signaling mutants. Pro35S:AtCKX lines, as well as a cytokinin receptor mutant ahk3, showed aberrations in the auxin response distribution in columella cells consistent with defects in the auxin transport machinery. Using in vivo real-time imaging of PIN3-GFP and PIN7-GFP in AtCKX3 overexpression and ahk3 backgrounds, we observed wild-type-like relocalization of PIN proteins in the columella early after gravistimulation, with gravity-induced relocalization of PIN7 faster than that of PIN3. Nonetheless, the cellular distribution of PIN3 and PIN7 and expression of PIN7 and the auxin influx carrier AUX1 was affected in AtCKX overexpression lines. Based on the retained cytokinin sensitivity in pin3 pin4 pin7 mutant, we propose the AUX1-mediated auxin transport rather than columella-located PIN proteins as a target of endogenous cytokinins in the control of root gravitropism. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Metabolic Engineering of Isoflavonoid Biosynthesis in Alfalfa1[w

PubMed Central

Deavours, Bettina E.; Dixon, Richard A.

2005-01-01

The potential health benefits of dietary isoflavones have generated considerable interest in engineering the synthesis of these phytoestrogens into plants. Genistein glucoside production (up to 50 nmol g−1 fresh weight) was engineered in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A (4′-O-methylgenistein) and pratensein (3′-hydroxybiochanin A) also accumulated. Although MtIFS1 was highly expressed in all organs examined, genistein accumulation was limited to leaves. MtIFS1-expressing lines accumulated several additional isoflavones, including formononetin and daidzein, in response to UV-B or Phoma medicaginis, whereas the chalcone and flavanone precursors of these compounds accumulated in control lines. Enhanced accumulation of the phytoalexin medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing plants. Microarray profiling indicated that MtIFS1 expression does not significantly alter global gene expression in the leaves. Our results highlight some of the challenges associated with metabolic engineering of plant natural products, including tissue-specific accumulation, potential for further modification by endogenous enzyme activities (hydroxylation, methylation, and glycosylation), and the differential response of engineered plants to environmental factors. PMID:16006598

Medroxyprogesterone acetate-treated human, primary endometrial epithelial cells reveal unique gene expression signature linked to innate immunity and HIV-1 susceptibility.

PubMed

Woods, Matthew W; Zahoor, Muhammad Atif; Dizzell, Sara; Verschoor, Chris P; Kaushic, Charu

2018-01-01

Medroxyprogesterone acetate (MPA), a progestin-based hormonal contraceptive designed to mimic progesterone, has been linked to increased human immunodeficiency virus (HIV-1) susceptibility. Genital epithelial cells (GECs) form the mucosal lining of the female genital tract (FGT) and provide the first line of protection against HIV-1. The impact of endogenous sex hormones or MPA on the gene expression profile of GECs has not been comprehensively documented. Using microarray analysis, we characterized the transcriptional profile of primary endometrial epithelial cells grown in physiological levels of E2, P4, and MPA. Each hormone treatment altered the gene expression profile of GECs in a unique manner. Interestingly, although MPA is a progestogen, the gene expression profile induced by it was distinct from P4. MPA increased gene expression of genes related to inflammation and cholesterol synthesis linked to innate immunity and HIV-1 susceptibility. The analysis of gene expression profiles provides insights into the effects of sex hormones and MPA on GECs and allows us to posit possible mechanisms of the MPA-mediated increase in HIV-1 acquisition. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A genome-wide resource for the analysis of protein localisation in Drosophila

PubMed Central

Sarov, Mihail; Barz, Christiane; Jambor, Helena; Hein, Marco Y; Schmied, Christopher; Suchold, Dana; Stender, Bettina; Janosch, Stephan; KJ, Vinay Vikas; Krishnan, RT; Krishnamoorthy, Aishwarya; Ferreira, Irene RS; Ejsmont, Radoslaw K; Finkl, Katja; Hasse, Susanne; Kämpfer, Philipp; Plewka, Nicole; Vinis, Elisabeth; Schloissnig, Siegfried; Knust, Elisabeth; Hartenstein, Volker; Mann, Matthias; Ramaswami, Mani; VijayRaghavan, K; Tomancak, Pavel; Schnorrer, Frank

2016-01-01

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts. DOI: http://dx.doi.org/10.7554/eLife.12068.001 PMID:26896675

CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models.

PubMed

Xue, Fuying; Wu, Yanping; Zhao, Xinghui; Zhao, Taoran; Meng, Ying; Zhao, Zhanzhong; Guo, Junwei; Chen, Wei

2016-08-01

Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca(2+) ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1. Copyright © 2016 Elsevier Inc. All rights reserved.

Hsa-mir-182 suppresses lung tumorigenesis through down regulation of RGS17 expression in vitro

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

Sun, Yihua; Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200031; Fang, Rong

2010-05-28

Lung cancer is one of the most devastating diseases worldwide. RGS17 is previously shown to be over-expressed in human lung adenocarcinomas and plays an important role in lung tumor growth. Here we have identified a miRNA, has-mir-182, involved in the regulation of RGS17 expression through two conserved sites located in its 3' UTR region. Consistently, endogenous RGS17 expression level is regulated by hsa-mir-182 in human lung cancer cell lines. Similar to the knockdown of RGS17, ectopic expression of hsa-mir-182 significantly inhibits lung cancer cell proliferation and anchorage-independent cell growth, which can be rescued by re-expression of RGS17. Taken together, thesemore » data have provided the first evidence of miRNA regulation of RGS17 expression in lung cancer.« less

Epigenetic modification of miR-141 regulates SKA2 by an endogenous ‘sponge’ HOTAIR in glioma

PubMed Central

Wang, Chao; Zong, Gang; Wang, Hong-Liang; Zhao, Bing

2016-01-01

Aberrant expression of miR-141 has recently implicated in the occurrence and development of various types of malignant tumors. However whether the involvement of miR-141 in the pathogenesis of glioma remains unknown. Here, we showed that miR-141 was markedly downregulated in glioma tissues and cell lines compared with normal brain tissues, and its expression correlated with the pathological grading. Enforced expression of miR-141 in glioma cells significantly inhibited cell proliferation, migration and invasion, whereas knockdown of miR-141 exerted opposite effect. Mechanistic investigations revealed that HOTAIR might act as an endogenous ‘sponge’ of miR-141, thereby regulating the derepression of SKA2. Further, we explored the molecular mechanism by which miR-141 expression was regulated, and found that the miR-141 promoter was hypermethylated and that promoter methylation of miR-141 was mediated by DNMT1 in glioma cells. Finally, both overexpression of miR-141 and knockdown of HOTAIR in a mouse model of human glioma resulted in significant reduction of tumor growth in vivo. Collectively, these results suggest that epigenetic modification of miR-141 and the interaction of ceRNA regulatory network will provide a new approach for therapeutics against glioma. PMID:27121316

Functional analysis of Discoidin domain receptor 2 mutation and expression in squamous cell lung cancer.

PubMed

Kobayashi-Watanabe, Naomi; Sato, Akemi; Watanabe, Tatsuro; Abe, Tomonori; Nakashima, Chiho; Sueoka, Eisaburo; Kimura, Shinya; Sueoka-Aragane, Naoko

2017-08-01

Discoidin domain receptor (DDR) 2 mutations have recently been reported to be candidate targets of molecular therapy in lung squamous cell carcinoma (SQCC). However, the status of DDR2 expression and mutations, as well as their precise roles in lung SQCC, have not been clarified. We here report DDR2 mutation and expression status in clinical samples and its role of lung SQCC. We investigated DDR2 expression and mutation status in 44 human clinical samples and 7 cell lines. Biological functions of DDR2 were assessed by in vitro cell invasion assay and animal model experiments. Endogenous DDR2 protein expression levels were high in one cell line, PC-1, and immunohistochemistry of lung cancer tissue array showed high levels of DDR2 protein in 29% of lung SQCC patients. A mutation (T681I) identified in lung SQCC and the cell line EBC-1 was detected among 44 primary lung SQCC samples and 7 lung SQCC cell lines. Although Forced expression of DDR2 and its mutant (T681I) led to induce SQCC cell invasion in vitro, only wild type DDR2 enhanced lung metastasis in an animal model. We also found that ectopic expression of DDR2 induced MMP-1 mRNA expression accompanied by phosphorylation of c-Jun after treatment with its ligand, collagen type I, but DDR2 with the T681I mutation did not, suggesting that T681I mutation is an inactivating mutation. Overexpression of DDR2 might contribute to tumor progression in lung SQCC. The overexpression of DDR2 could be potential molecular target of lung SQCC. Copyright © 2017 Elsevier B.V. All rights reserved.

Gtl2lacZ, an insertional mutation on mouse chromosome 12 with parental origin-dependent phenotype.

PubMed

Schuster-Gossler, K; Simon-Chazottes, D; Guenet, J L; Zachgo, J; Gossler, A

1996-01-01

We have produced a transgenic mouse line, Gtl2lacZ (Gene trap locus 2), that carries an insertional mutation with a dominant modified pattern of inheritance:heterozygous Gtl2lacZ mice that inherited the transgene from the father show a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype is strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. On a mixed genetic background this pattern of inheritance was reversible upon transmission of the transgene through the germ line of the opposite sex. On a predominantly 129/Sv genetic background, however, transgene passage through the female germ line modified the transgene effect, such that the penetrance of the mutation was drastically reduced and the phenotype was no longer obvious after subsequent male germ line transmission. Expression of the transgene, however, was neither affected by genetic background nor by parental legacy. Gtl2lacZ maps to mouse Chromosome 12 in a region that displays imprinting effects associated with maternal and paternal disomy. Our results suggest that the transgene insertion in Gtl2lacZ mice affects an endogenous gene(s) required for fetal and postnatal growth and that this gene(s) is predominantly paternally expressed.

Expression of C-terminal deleted p53 isoforms in neuroblastoma

PubMed Central

Goldschneider, David; Horvilleur, Emilie; Plassa, Louis-François; Guillaud-Bataille, Marine; Million, Karine; Wittmer-Dupret, Evelyne; Danglot, Gisèle; de Thé, Hughes; Bénard, Jean; May, Evelyne; Douc-Rasy, Sétha

2006-01-01

The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53β isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122–2137]. Our results show, for the first time, that the p53β isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development. PMID:17028100

DREAM regulates BDNF-dependent spinal sensitization

PubMed Central

2010-01-01

Background The transcriptional repressor DREAM (downstream regulatory element antagonist modulator) controls the expression of prodynorphin and has been involved in the modulation of endogenous responses to pain. To investigate the role of DREAM in central mechanisms of pain sensitization, we used a line of transgenic mice (L1) overexpressing a Ca2+- and cAMP-insensitive DREAM mutant in spinal cord and dorsal root ganglia. Results L1 DREAM transgenic mice showed reduced expression in the spinal cord of several genes related to pain, including prodynorphin and BDNF (brain-derived neurotrophic factor) and a state of basal hyperalgesia without change in A-type currents. Peripheral inflammation produced enhancement of spinal reflexes and increased expression of BDNF in wild type but not in DREAM transgenic mice. The enhancement of the spinal reflexes was reproduced in vitro by persistent electrical stimulation of C-fibers in wild type but not in transgenic mice. Exposure to exogenous BDNF produced a long-term enhancement of dorsal root-ventral root responses in transgenic mice. Conclusions Our results indicate that endogenous BDNF is involved in spinal sensitization following inflammation and that blockade of BDNF induction in DREAM transgenic mice underlies the failure to develop spinal sensitization. PMID:21167062

Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines.

PubMed

Meisenberg, Annika; Kaschuba, Dagmar; Balfanz, Sabine; Jordan, Nadine; Baumann, Arnd

2015-10-01

Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses. Copyright © 2015 Elsevier Inc. All rights reserved.

The Role of SncN and Ski in Mammary Epithelial Cell Transformation

DTIC Science & Technology

2005-07-01

cellular activities through the Smad proteins. Upon phosphorylation by the active TGFβ receptor kinases, Smad2 and Smad3 oligomerize with Smad4, translocate...sequence in sense and antisense orientation with an intervening linker . Primer pairs were designed to generate single-strand overhangs upon annealing...nuclear protein, based on studies of ectopically expressed SnoN and endogenous SnoN in cancer cell lines. In the nucleus, SnoN binds to Smad2, Smad3 , and

Identifying p53 Transactivation Domain 1-Specific Inhibitors to Alleviate the Side Effects of Prostate Cancer Therapy

DTIC Science & Technology

2014-10-01

harnessed the latest genome editing tools of the CRISPR /Cas9 (Clustered Regulatory Interspaced Short Palindromic Repeats) system to generate...immortalized MEF reporter lines that stably express reporters under the control of the endogenous genes. The CRISPR /Cas9 system, which was recently developed...cells carrying different retroviral reporter constructs. Thus, even though using the CRISPR /Cas9 system is inherently a longer process, the use of

Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system.

PubMed

Feng, Yong; Sassi, Slim; Shen, Jacson K; Yang, Xiaoqian; Gao, Yan; Osaka, Eiji; Zhang, Jianming; Yang, Shuhua; Yang, Cao; Mankin, Henry J; Hornicek, Francis J; Duan, Zhenfeng

2015-02-01

Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

NASA Astrophysics Data System (ADS)

Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

2017-05-01

The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

Identification, characterization, and comparative genomic distribution of the HERV-K (HML-2) group of human endogenous retroviruses

PubMed Central

2011-01-01

Background Integration of retroviral DNA into a germ cell may lead to a provirus that is transmitted vertically to that host's offspring as an endogenous retrovirus (ERV). In humans, ERVs (HERVs) comprise about 8% of the genome, the vast majority of which are truncated and/or highly mutated and no longer encode functional genes. The most recently active retroviruses that integrated into the human germ line are members of the Betaretrovirus-like HERV-K (HML-2) group, many of which contain intact open reading frames (ORFs) in some or all genes, sometimes encoding functional proteins that are expressed in various tissues. Interestingly, this expression is upregulated in many tumors ranging from breast and ovarian tissues to lymphomas and melanomas, as well as schizophrenia, rheumatoid arthritis, and other disorders. Results No study to date has characterized all HML-2 elements in the genome, an essential step towards determining a possible functional role of HML-2 expression in disease. We present here the most comprehensive and accurate catalog of all full-length and partial HML-2 proviruses, as well as solo LTR elements, within the published human genome to date. Furthermore, we provide evidence for preferential maintenance of proviruses and solo LTR elements on gene-rich chromosomes of the human genome and in proximity to gene regions. Conclusions Our analysis has found and corrected several errors in the annotation of HML-2 elements in the human genome, including mislabeling of a newly identified group called HML-11. HML-elements have been implicated in a wide array of diseases, and characterization of these elements will play a fundamental role to understand the relationship between endogenous retrovirus expression and disease. PMID:22067224

SEREX analysis for tumor antigen identification in a mouse model of adenocarcinoma.

PubMed

Hampton, T A; Conry, R M; Khazaeli, M B; Shaw, D R; Curiel, D T; LoBuglio, A F; Strong, T V

2000-03-01

Evaluation of immunotherapy strategies in mouse models of carcinoma is hampered by the limited number of known murine tumor antigens (Ags). Although tumor Ags can be identified based on cytotoxic T-cell activation, this approach is not readily accomplished for many tumor types. We applied an alternative strategy based on a humoral immune response, SEREX, to the identification of tumor Ags in the murine colon adenocarcinoma cell line MC38. Immunization of syngeneic C57BL/6 mice with MC38 cells by three different methods induced a protective immune response with concomitant production of anti-MC38 antibodies. Immunoscreening of an MC38-derived expression library resulted in the identification of the endogenous ecotropic leukemia virus envelope (env) protein and the murine ATRX protein as candidate tumor Ags. Northern blot analysis demonstrated high levels of expression of the env transcript in MC38 cells and in several other murine tumor cell lines, whereas expression in normal colonic epithelium was absent. ATRX was found to be variably expressed in tumor cell lines and in normal tissue. Further analysis of the expressed env sequence indicated that it represents a nonmutated tumor Ag. Polynucleotide immunization with DNA encoding the env polypeptide resulted in strong and specific antibody responses to this self Ag in all immunized mice. Thus, SEREX offers a rapid means of identifying tumor Ags in murine cancer models.

The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish

PubMed Central

Hall, Chris; Flores, Maria Vega; Storm, Thilo; Crosier, Kathy; Crosier, Phil

2007-01-01

Background How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable. Results We used regulatory regions of the zebrafish lysozyme C (lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria. Conclusion These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation. PMID:17477879

MicroRNA858 Is a Potential Regulator of Phenylpropanoid Pathway and Plant Development1

PubMed Central

Sharma, Deepika; Tiwari, Manish; Pandey, Ashutosh; Bhatia, Chitra; Sharma, Ashish; Trivedi, Prabodh Kumar

2016-01-01

MicroRNAs (miRNAs) are endogenous, noncoding small RNAs that function as critical regulators of gene expression. In plants, miRNAs have shown their potential as regulators of growth, development, signal transduction, and stress tolerance. Although the miRNA-mediated regulation of several processes is known, the involvement of miRNAs in regulating secondary plant product biosynthesis is poorly understood. In this study, we functionally characterized Arabidopsis (Arabidopsis thaliana) miR858a, which putatively targets R2R3-MYB transcription factors involved in flavonoid biosynthesis. Overexpression of miR858a in Arabidopsis led to the down-regulation of several MYB transcription factors regulating flavonoid biosynthesis. In contrast to the robust growth and early flowering of miR858OX plants, reduction of plant growth and delayed flowering were observed in Arabidopsis transgenic lines expressing an artificial miRNA target mimic (MIM858). Genome-wide expression analysis using transgenic lines suggested that miR858a targets a number of regulatory factors that modulate the expression of downstream genes involved in plant development and hormonal and stress responses. Furthermore, higher expression of MYBs in MIM858 lines leads to redirection of the metabolic flux towards the synthesis of flavonoids at the cost of lignin synthesis. Altogether, our study has established the potential role of light-regulated miR858a in flavonoid biosynthesis and plant growth and development. PMID:27208307

Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

PubMed Central

Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

2016-01-01

The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.

PubMed

Wert, S E; Glasser, S W; Korfhagen, T R; Whitsett, J A

1993-04-01

Transgenic animals bearing a chimeric gene containing 5'-flanking regions of the human surfactant protein C (SP-C) gene ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene were analyzed by in situ hybridization histochemistry to determine the temporal and spatial distribution of transgene expression during organogenesis of the murine lung. Ontogenic expression of the SP-C-CAT gene was compared to that of the endogenous SP-C gene and to the Clara cell CC10 gene. High levels of SP-C-CAT expression were observed as early as Day 10 of gestation in epithelial cells of the primordial lung buds. Low levels of endogenous SP-C mRNA were detected a day later, but only in the more distal epithelial cells of the newly formed, primitive, lobar bronchi. On Gestational Days 13 through 16, transcripts for both the endogenous and chimeric gene were restricted to distal epithelial elements of the branching bronchial tubules and were no longer detected in the more proximal regions of the bronchial tree. Although high levels of SP-C-CAT expression were maintained throughout organogenesis, endogenous SP-C expression increased dramatically on Gestational Day 15, coincident with acinar tubule differentiation at the lung periphery. Low levels of endogenous CC10 expression were detected by Gestational Day 16 in both lobar and segmental bronchi. By the time of birth, CC10 transcripts were expressed at high levels in the trachea and at all levels of the bronchial tree; endogenous SP-C mRNA was restricted to epithelial cells of the terminal alveolar saccules; and SP-C-CAT expression was now detected in both alveolar and bronchiolar epithelial cells. These results indicate that (1) cis-acting regulatory elements of the human SP-C gene can direct high levels of foreign gene expression to epithelial cells of the embryonic mouse lung; (2) expression of the human SP-C-CAT chimeric gene is developmentally regulated, exhibiting a morphogenic expression pattern similar, but not identical, to that of the endogenous murine SP-C gene; (3) the embryonic expression of endogenous SP-C and chimeric SP-C-CAT transcripts identifies progenitor cells of the distal respiratory epithelium; and (4) differentiation of bronchial epithelium is coincident with loss of SP-C expression and subsequent acquisition of CC10 expression in proximal regions of the developing bronchial tubules.

Ionizing radiation modulates the surface expression of human leukocyte antigen-G in a human melanoma cell line.

PubMed

Michelin, Severino; Gallegos, Cristina E; Dubner, Diana; Favier, Benoit; Carosella, Edgardo D

2009-12-01

Human leukocyte antigen G (HLA-G) is a nonclassical HLA class I molecule involved in fetus protection from the maternal immune system, transplant tolerance, and viral and tumoral immune escape. Tumor-specific HLA-G expression has been described for a wide variety of malignancies, including melanomas. The aim of this study was to evaluate whether ionizing radiation (IR) could modulate the surface expression of HLA-G1 in a human melanoma cell line that expresses endogenously membrane-bound HLA-G1. For this purpose, cells were exposed to increasing doses of gamma-irradiation (0-20 Gy) and HLA-G1 levels at the plasma membrane were analyzed at different times postirradiation by flow cytometry. HLA-G total expression and the presence of the soluble form of HLA-G1 (sHLA-G1) in the culture medium of irradiated cells were also evaluated. IR was capable of downregulating cell surface and total HLA-G levels, with a concomitant increase of sHLA-G1 in the medium. These results could indicate that gamma-irradiation decreases HLA-G1 surface levels by enhancing the proteolytic cleavage of this molecule.

Inhibition of connective tissue growth factor (CTGF/CCN2) in gallbladder cancer cells leads to decreased growth in vitro

PubMed Central

Garcia, Patricia; Leal, Pamela; Ili, Carmen; Brebi, Priscilla; Alvarez, Hector; Roa, Juan C

2013-01-01

Gallbladder cancer (GBC) is an aggressive neoplasm associated with late diagnosis, unsatisfactory treatment and poor prognosis. Previous work showed that connective tissue growth factor (CTGF) expression is increased in this malignancy. This matricellular protein plays an important role in various cellular processes and its involvement in the tumorigenesis of several human cancers has been demonstrated. However, the precise function of CTGF expression in cancer cells is yet to be determined. The aim of this study was to evaluate the CTGF expression in gallbladder cancer cell lines, and its effect on cell viability, colony formation and in vitro cell migration. CTGF expression was evaluated in seven GBC cell lines by Western blot assay. Endogenous CTGF expression was downregulated by lentiviral shRNA directed against CTGF mRNA in G-415 cells, and the effects on cell viability, anchorage-independent growth and migration was assessed by comparing them to scrambled vector-transfected cells. Knockdown of CTGF resulted in significant reduction in cell viability, colony formation and anchorage-independent growth (P < 0.05). An increased p27 expression was observed in G-415 cells with loss of CTGF function. Our results suggest that high expression of this protein in gallbladder cancer may confer a growth advantage for neoplastic cells. PMID:23593935

Development of an endogenous virus-free line of chickens susceptible to all subgroups of avian leukosis virus.

PubMed

Zhang, Huanmin; Bacon, Larry D; Fadly, Aly M

2008-09-01

Primary chicken embryo fibroblasts (CEF) from special specific pathogen-free chicken lines are used for detection of contamination of adult or embryonic tissues, meconium, or tissue culture fluids with avian leukosis viruses (ALV). The suitability and efficiency of such tests depend on the susceptibility of CEF to the various subgroups of exogenous as well as endogenous ALV. The ideal CEF for such tests should be not only susceptible to all retroviruses, but also free of endogenous viruses so that such tests are immune to any interference that may occur between the endogenous and the tested (exogenous) viruses. CEF and/or chickens free of endogenous viruses are also desirable for gene transfer studies using retroviral vectors, such as RNA interference (RNAi) experiments and transgenic work. The absence of ev genes in CEF or chickens can empower clean detection of successful RNAi construct delivery or gene transfer. CEF free of ev genes are also essential reagents routinely used in growing and detecting unknown retroviruses in varied viral assays. This report documents the development of a new line of chickens, 0.TVB*S1, that is free of endogenous viruses and susceptible to all subgroups of ALV identified in chickens.

ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer

PubMed Central

2012-01-01

Background ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transformed urothelial cells. Results It was shown that in the renal system both the non-glycosylated and glycosylated form of ZIP8 was expressed in the proximal tubule cells with localization of ZIP8 to the cytoplasm and cell membrane; findings in line with previous studies on ZIP8. The studies in the bladder were the first to show that ZIP8 was expressed in normal urothelium and that ZIP8 could be localized to the paranuclear region. Studies in the UROtsa cell line confirmed a paranuclear localization of ZIP8, however addition of growth medium to the cells increased the expression of the protein in the UROtsa cells. In archival human samples of the normal urothelium, the expression of ZIP8 was variable in intensity whereas in urothelial cancers ZIP8 was expressed in 13 of 14 samples, with one high grade invasive urothelial cancer showing no expression. The expression of ZIP8 was similar in the Cd+2 and As+3 transformed UROtsa cell lines and their tumor transplants. Conclusion This is the first study which shows that ZIP8 is expressed in the normal urothelium and in bladder cancer. In addition the normal UROtsa cell line and its transformed counterparts show similar expression of ZIP8 compared to the normal urothelium and the urothelial cancers suggesting that the UROtsa cell line could serve as a model system to study the expression of ZIP8 in bladder disease. PMID:22550998

Expression of the pol gene of human endogenous retroviruses HERV-K and -W in leukemia patients.

PubMed

Bergallo, Massimiliano; Montanari, Paola; Mareschi, Katia; Merlino, Chiara; Berger, Massimo; Bini, Ilaria; Daprà, Valentina; Galliano, Ilaria; Fagioli, Franca

2017-12-01

The human endogenous retroviruses (HERVs) are a family of endogenous retroviruses that integrated into the germ cell DNA of primates over 30 million years ago. HERV expression seems impaired in several diseases, ranging from autoimmune to neoplastic disorders. The purpose of this study was to evaluate the overall endogenous retroviral transcription profile in bone marrow (BM) samples. A total of 30 paediatric high-risk leukaemia patients (lymphoid and myeloid malignancies) were tested for HERVs virus gene expression. Our findings show that HERV-K expression was significantly higher in leukaemia patients when compared to healthy donors of a similar median age. We observed a significantly high expression of HERV-K in acute lymphoblastic leukemia (ALL) patients. In this study, we also found a relative overexpression of the endogenous retrovirus HERV-K in BM cells from the majority of leukemia samples analyzed, in particular in ALL. This overexpression might be related to lymphatic leukemogenesis and it warrants further investigations.

The Antitumor Effect of C-terminus of Hsp70-Interacting Protein via Degradation of c-Met in Small Cell Lung Cancer.

PubMed

Cho, Sung Ho; Kim, Jong In; Kim, Hyun Su; Park, Sung Dal; Jang, Kang Won

2017-06-01

The mesenchymal-epithelial transition factor (MET) receptor can be overexpressed in solid tumors, including small cell lung cancer (SCLC). However, the molecular mechanism regulating MET stability and turnover in SCLC remains undefined. One potential mechanism of MET regulation involves the C-terminus of Hsp70-interacting protein (CHIP), which targets heat shock protein 90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, we investigated the functional effects of CHIP expression on MET regulation and the control of SCLC cell apoptosis and invasion. To evaluate the expression of CHIP and c-Met, which is a protein that in humans is encoded by the MET gene (the MET proto-oncogene), we examined the expression pattern of c-Met and CHIP in SCLC cell lines by western blotting. To investigate whether CHIP overexpression reduced cell proliferation and invasive activity in SCLC cell lines, we transfected cells with CHIP and performed a cell viability assay and cellular apoptosis assays. We found an inverse relationship between the expression of CHIP and MET in SCLC cell lines (n=5). CHIP destabilized the endogenous MET receptor in SCLC cell lines, indicating an essential role for CHIP in the regulation of MET degradation. In addition, CHIP inhibited MET-dependent pathways, and invasion, cell growth, and apoptosis were reduced by CHIP overexpression in SCLC cell lines. CHIP is capable of regulating SCLC cell apoptosis and invasion by inhibiting MET-mediated cytoskeletal and cell survival pathways in NCI-H69 cells. CHIP suppresses MET-dependent signaling, and regulates MET-mediated SCLC motility.

Tumor suppressor BRCA1 is expressed in prostate cancer and controls IGF-I receptor (IGF-IR) gene transcription in an androgen receptor-dependent manner

PubMed Central

Schayek, Hagit; Haugk, Kathy; Sun, Shihua; True, Lawrence D.; Plymate, Stephen R.; Werner, Haim

2010-01-01

Purpose The insulin-like growth factor (IGF) system plays an important role in prostate cancer. The BRCA1 gene encodes a transcription factor with tumor suppressor activity. The involvement of BRCA1 in prostate cancer, however, has not yet been elucidated. The purpose of the present study was to examine the functional correlations between BRCA1 and the IGF system in prostate cancer. Experimental Design An immunohistochemical analysis of BRCA1 was performed on Tissue Microarrays comprising 203 primary prostate cancer specimens. In addition, BRCA1 levels were measured in prostate cancer xenografts and in cell lines representing early stages of the disease (P69 cells) and advanced stages (M12 cells). The ability of BRCA1 to regulate IGF-IR expression was studied by coexpression experiments using a BRCA1 expression vector along with an IGF-IR promoter-luciferase reporter. Results We found significantly elevated BRCA1 levels in prostate cancer in comparison to histologically normal prostate tissue (p < 0.001). In addition, an inverse correlation between BRCA1 and IGF-IR levels was observed in the AR-negative P69 and M12 prostate cancer-derived cell lines. Coexpression experiments in M12 cells revealed that BRCA1 was able to suppress IGF-IR promoter activity and endogenous IGF-IR levels. On the other hand, BRCA1 enhanced IGF-IR levels in LnCaP C4-2 cells expressing an endogenous AR. Conclusions We provide evidence that BRCA1 differentially regulates IGF-IR expression in AR positive and negative prostate cancer cells. The mechanism of action of BRCA1 involves modulation of IGF-IR gene transcription. In addition, immunohistochemical data is consistent with a potential survival role of BRCA1 in prostate cancer. PMID:19223505

p21Waf1/Cip1/Sdi1 Prevents Apoptosis as Well as Stimulates Growth in Cells Transformed or Immortalized by Human T-Cell Leukemia Virus Type 1-Encoded Tax

PubMed Central

Kawata, Sanae; Ariumi, Yasuo; Shimotohno, Kunitada

2003-01-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21Waf1/Cip1/Sdi1, resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-κB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms. PMID:12805427

An alternative approach in regulation of expression of a transgene by endogenous miR-145 in carcinoma and normal breast cell lines.

PubMed

Ghanbari Safari, Maryam; Baesi, Kazem; Hosseinkhani, Saman

2017-03-01

MicroRNAs are small noncoding RNAs that regulate gene expression by repressing translation of target cellular transcripts. Increasing evidences indicate that miRNAs have different expression profiles and play crucial roles in numerous cellular processes. Delivery and expression of transgenes for cancer therapy must be specific for tumors to avoid killing of healthy tissues. Many investigators have shown that transgene expression can be suppressed in normal cells using vectors that are responsive to microRNA regulation. To overcome this problem, miR-145 that exhibits downregulation in many types of cancer cells was chosen for posttranscriptional regulatory systems mediated by microRNAs. In this study, a psiCHECK-145T vector carrying four tandem copies of target sequences of miR-145 into 3'-UTR of the Renilla luciferase gene was constructed. Renilla luciferase activity from the psiCHECK-145T vector was 57% lower in MCF10A cells with high miR-145 expression as compared to a control condition. Additionally, overexpression of miR-145 in MCF-7 cells with low expression level of miR-145 showed more than 76% reduction in the Renilla luciferase activity from the psiCHECK-145T vector. Inclusion of miR-145 target sequences into the 3'-UTR of the Renilla luciferase gene is a feasible strategy for restricting transgene expression in a breast cancer cell line while sparing a breast normal cell line. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

The Endogenous GRP78 Interactome in Human Head and Neck Cancers: A Deterministic Role of Cell Surface GRP78 in Cancer Stemness.

PubMed

Chen, Hsin-Ying; Chang, Joseph Tung-Chieh; Chien, Kun-Yi; Lee, Yun-Shien; You, Guo-Rung; Cheng, Ann-Joy

2018-01-11

Cell surface glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, was suggested to be a cancer stem cell marker, but the influence of this molecule on cancer stemness is poorly characterized. In this study, we developed a mass spectrometry platform to detect the endogenous interactome of GRP78 and investigated its role in cancer stemness. The interactome results showed that cell surface GRP78 associates with multiple molecules. The influence of cell population heterogeneity of head and neck cancer cell lines (OECM1, FaDu, and BM2) according to the cell surface expression levels of GRP78 and the GRP78 interactome protein, Progranulin, was investigated. The four sorted cell groups exhibited distinct cell cycle distributions, asymmetric/symmetric cell divisions, and different relative expression levels of stemness markers. Our results demonstrate that cell surface GRP78 promotes cancer stemness, whereas drives cells toward a non-stemlike phenotype when it chaperones Progranulin. We conclude that cell surface GRP78 is a chaperone exerting a deterministic influence on cancer stemness.

The lymphotoxin promoter is stimulated by HTLV-I tax activation of NF-kappa B in human T-cell lines.

PubMed

Paul, N L; Millet, I; Ruddle, N H

1993-07-01

The HTLV-I transcriptional activator tax was used to gain insight into the mechanism of lymphotoxin (LT; TNF-beta) gene induction. Tax-expressing cell lines produce LT biologic activity. An LT promoter (LT-293) CAT construct that contained an NF-kappa B site was active in the LT-producing C81-66-45 cell line, which contains defective HTLV-I but expresses tax. The observation that a mutated LT-kappa B construct (M1-CAT) was inactive in C81-66-45, confirmed the importance of NF-kappa B in LT gene expression. Tax was transfected into HTLV-I-negative human T-cell lines. Jurkat T cells stably expressing tax contained elevated levels of NF-kappa B that directly bound to the LT-kappa B site. Tax co-transfected with reporter constructs into Jurkat cells maximally activated HTLV-I-LTR-CAT and kappa B-fos-CAT and also activated LT-293 to a lesser extent. In JM T cells, tax induced LT-293 activity by two- to four-fold, though there was no induction of M1-CAT. The increase in LT-293 CAT activity mirrored the increase in LT biologic activity seen under these conditions. These studies, the first to demonstrate induction of LT promoter activity over basal levels, indicate that HTLV-I tax causes low-level activation of both endogenous LT and the LT promoter, at least in part through activation of NF-kappa B.

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity

PubMed Central

Chittaranjan, Suganthi; Chan, Susanna; Yang, Cindy; Yang, Kevin C.; Chen, Vincent; Moradian, Annie; Firme, Marlo; Song, Jungeun; Go, Nancy E.; Blough, Michael D.; Chan, Jennifer A.; Cairncross, J. Gregory; Gorski, Sharon M.; Morin, Gregg B.; Yip, Stephen; Marra, Marco A.

2014-01-01

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism of reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples. PMID:25277207

Upregulating endogenous genes by an RNA-programmable artificial transactivator

PubMed Central

Fimiani, Cristina; Goina, Elisa; Mallamaci, Antonello

2015-01-01

To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action. PMID:26152305

Involvement of overexpressed wild-type BRAF in the growth of malignant melanoma cell lines.

PubMed

Tanami, Hideaki; Imoto, Issei; Hirasawa, Akira; Yuki, Yasuhiro; Sonoda, Itaru; Inoue, Jun; Yasui, Kohichiro; Misawa-Furihata, Akiko; Kawakami, Yutaka; Inazawa, Johji

2004-11-18

Comparative genomic hybridization (CGH) using 40 cell lines derived from malignant melanomas (MMs) revealed frequent amplification at 7q33-q34 containing BRAF gene, which often is mutated in MM. We found this gene to be amplified to a remarkable degree in the MM cell lines that exhibited high-level gains at 7q33-q34 in CGH. Among 40 cell lines, the eight lines that revealed neither BRAF nor NRAS mutations showed even higher levels of BRAF mRNA expression than the 32 mutated lines, although DNA amplification at 7q33-q34 was not detected in every lines overexpressing BRAF. MM cells that carried wild-type BRAF and NRAS showed constitutive overexpression of B-Raf protein and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), even after serum starvation. Not only downregulation of the endogenously overexpressed wild-type B-Raf by antisense oligonucleotide but also a treatment with an inhibitor of mitogen-activated protein kinase kinase (MAPKK, MEK) reduced phosphorylated ERK1/2 and cell growth, whereas the exogenously expressed wild-type B-Raf promoted cell growth in MM cells. Our results provide the evidence that overexpression of wild-type B-Raf, in part but not always as a result of gene amplification, is one of the mechanisms underlying constitutive activation of the MAPK pathway that stimulates growth of MM cells.

Protection of dopaminergic neurons by electroconvulsive shock in an animal model of Parkinson's disease.

PubMed

Anastasia, Agustín; de Erausquin, Gabriel A; Wojnacki, José; Mascó, Daniel H

2007-11-01

Electroconvulsive shock (ECS) improves motor function in Parkinson's disease. In rats, ECS stimulates the expression of various factors some of which have been proposed to exert neuroprotective actions. We have investigated the effects of ECS on 6-hydroxydopamine (6-OHDA)-injected rats. Three weeks after a unilateral administration of 6-OHDA, 85-95% nigral dopaminergic neurons are lost. Chronic ECS prevented this cell loss, protect the nigrostriatal pathway (assessed by FloroGold retrograde labeling) and reduce motor impairment in 6-OHDA-treated animals. Injection of 6-OHDA caused loss of expression of glial cell-line derived neurotrophic factor (GDNF) in the substantia nigra. Chronic ECS completely prevented this loss of GDNF expression in 6-OHDA-treated animals. We also found that protected dopaminergic neurons co-express GDNF receptor proteins. These results strongly suggest that endogenous changes in GDNF expression may participate in the neuroprotective mechanism of ECS against 6-OHDA induced toxicity.

A heterologous hormone response element enhances expression of rat beta-casein promoter-driven chloramphenicol acetyltransferase fusion genes in the mammary gland of transgenic mice.

PubMed

Greenberg, N M; Reding, T V; Duffy, T; Rosen, J M

1991-10-01

Previous studies have demonstrated that the entire rat beta-casein (R beta C) gene and a -524/+490 R beta C fragment-chloramphenicol acetyltransferase (CAT) fusion gene are expressed preferentially in the mammary gland of transgenic mice in a developmentally regulated fashion. However, transgene expression was infrequent, less than 1% of that observed for the endogenous gene, and varied as much as 500-fold, presumably due to the site of chromosomal integration. To determine whether a heterologous hormone-responsive enhancer could be used to increase both the level and frequency of expression in the mammary gland, a fragment derived from the mouse mammary tumor virus long terminal repeat containing four hormone response elements (HREs) was inserted into the R beta C promoter at a site not known to contain transcriptional regulatory elements. Transgenic mice generated which carried HRE-enhanced R beta C-CAT fusion genes expressed CAT activity in the mammary glands of all founder lines examined at levels that were on average 13-fold greater than for lines generated with similar constructs not carrying HREs. In the highest expressing line, the level of HRE-enhanced transgene expression was found to be developmentally regulated, increasing 14-fold in the mammary gland from virgin to day 10 of lactation. In this line, expression was also observed in the thymus and spleen; however, the level of CAT activity was 4-fold lower than in the mammary gland and was not developmentally regulated. In adrenalectomized mice, the administration of dexamethasone stimulated CAT expression in the mammary gland but not in the thymus and spleen. These studies demonstrate that in the context of the R beta C promoter, the HRE functions in the mammary gland to increase both the frequency and level of transgene expression.

Obesity resistant mechanisms in the Lean polygenic mouse model as indicated by liver transcriptome and expression of selected genes in skeletal muscle

PubMed Central

2011-01-01

Background Divergently selected Lean and Fat mouse lines represent unique models for a polygenic form of resistance and susceptibility to obesity development. Previous research on these lines focused mainly on obesity-susceptible factors in the Fat line. This study aimed to examine the molecular basis of obesity-resistant mechanisms in the Lean line by analyzing various fat depots and organs, the liver transcriptome of selected metabolic pathways, plasma and lipid homeostasis and expression of selected skeletal muscle genes. Results Expression profiling using our custom Steroltalk v2 microarray demonstrated that Lean mice exhibit a higher hepatic expression of cholesterol biosynthesis genes compared to the Fat line, although this was not reflected in elevation of total plasma or liver cholesterol. However, FPLC analysis showed that protective HDL cholesterol was elevated in Lean mice. A significant difference between the strains was also found in bile acid metabolism. Lean mice had a higher expression of Cyp8b1, a regulatory enzyme of bile acid synthesis, and the Abcb11 bile acid transporter gene responsible for export of acids to the bile. Additionally, a higher content of blood circulating bile acids was observed in Lean mice. Elevated HDL and upregulation of some bile acids synthesis and transport genes suggests enhanced reverse cholesterol transport in the Lean line - the flux of cholesterol out of the body is higher which is compensated by upregulation of endogenous cholesterol biosynthesis. Increased skeletal muscle Il6 and Dio2 mRNA levels as well as increased activity of muscle succinic acid dehydrogenase (SDH) in the Lean mice demonstrates for the first time that changes in muscle energy metabolism play important role in the Lean line phenotype determination and corroborate our previous findings of increased physical activity and thermogenesis in this line. Finally, differential expression of Abcb11 and Dio2 identifies novel strong positional candidate genes as they map within the quantitative trait loci (QTL) regions detected previously in crosses between the Lean and Fat mice. Conclusion We identified novel candidate molecular targets and metabolic changes which can at least in part explain resistance to obesity development in the Lean line. The major difference between the Lean and Fat mice was in increased liver cholesterol biosynthesis gene mRNA expression, bile acid metabolism and changes in selected muscle genes' expression in the Lean line. The liver Abcb11 and muscle Dio2 were identified as novel positional candidate genes to explain part of the phenotypic difference between the Lean and Fat lines. PMID:21291556

Functional characterization of three MicroRNAs of the Asian Tiger Mosquito, Aedes albopictus

PubMed Central

2013-01-01

Background Temporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes. Methods miRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes. Results The knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults. Conclusion This study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector. PMID:23924583

MALAT1-miR-124-RBG2 axis is involved in growth and invasion of HR-HPV-positive cervical cancer cells.

PubMed

Liu, Shikai; Song, Lili; Zeng, Saitian; Zhang, Liang

2016-01-01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT 1) is a large, infrequently spliced non-coding RNA aberrantly expressed in cervical cancer. But the molecular mechanisms of its oncogenic role are still not quite clear. The present study explored whether there is a competing endogenous RNAs (ceRNAs) mechanism involved in the oncogenic effect of MALAT1. MALAT1 expression was firstly verified in high-risk human papillomavirus (HR-HPV)-positive tumor tissues and cell lines. Its regulation over miR-124 and the downstream target of miR-124 in regulation of growth, invasion, and apoptosis of the cancer cells are also studied. Findings of this study confirmed higher MALAT1 expression in HR-HPV (+) cervical cancer. Knockdown of endogenous MALAT1 significantly reduced cell growth rate and invasion and increased cell apoptosis of Hela and siHa cells. Besides, knockdown of MALAT1 increased the expression of miRNA-124, while ectopic expression of miR-124 decreased MALAT1 expression. In addition, we also verified a direct interaction between miR-124 and 3'UTR of GRB2. MALAT1 can indirectly modulate GRB2 expression via competing miR-124. Knockdown of GRB2 reduced cell invasion and increased cell apoptosis. In conclusion, MALAT1 can promote HR-HPV (+) cancer cell growth and invasion at least partially through the MALAT1-miR-124-RBG2 axis. This finding might provide some useful evidence about the lncRNA interaction regulatory network in tumorigenesis cervical cancer.

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells

PubMed Central

Fang, Feng; Zheng, Jiamao; Galbaugh, Traci L; Fiorillo, Alyson A; Hjort, Elizabeth E; Zeng, Xianke; Clevenger, Charles V

2010-01-01

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB knockdown with two independent siRNAs was shown to impair PRL-induced reporter expression in breast cancer cell line. cDNA microarray analysis was performed on these cells to assess the effect of CypB reduction, and revealed a significant decrease in PRL-induced endogenous gene expression in two breast cancer cell lines. Parallel functional assays revealed corresponding alterations of both anchorage-independent cell growth and cell motility of breast cancer cells. Our results demonstrate that CypB expression levels significantly modulate PRL-induced function in breast cancer cells ultimately resulting in enhanced levels of PRL-responsive gene expression, cell growth, and migration. Given the increasingly appreciated role of PRL in the pathogenesis of breast cancer, the actions of CypB detailed here are of biological significance. PMID:20237142

Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells.

PubMed

Fang, Feng; Zheng, Jiamao; Galbaugh, Traci L; Fiorillo, Alyson A; Hjort, Elizabeth E; Zeng, Xianke; Clevenger, Charles V

2010-06-01

The effects of prolactin (PRL) during the pathogenesis of breast cancer are mediated in part though Stat5 activity enhanced by its interaction with its transcriptional inducer, the prolyl isomerase cyclophilin B (CypB). We have demonstrated that knockdown of CypB decreases cell growth, proliferation, and migration, and CypB expression is associated with malignant progression of breast cancer. In this study, we examined the effect of CypB knockdown on PRL signaling in breast cancer cells. CypB knockdown with two independent siRNAs was shown to impair PRL-induced reporter expression in breast cancer cell line. cDNA microarray analysis was performed on these cells to assess the effect of CypB reduction, and revealed a significant decrease in PRL-induced endogenous gene expression in two breast cancer cell lines. Parallel functional assays revealed corresponding alterations of both anchorage-independent cell growth and cell motility of breast cancer cells. Our results demonstrate that CypB expression levels significantly modulate PRL-induced function in breast cancer cells ultimately resulting in enhanced levels of PRL-responsive gene expression, cell growth, and migration. Given the increasingly appreciated role of PRL in the pathogenesis of breast cancer, the actions of CypB detailed here are of biological significance.

Functional analysis of the DEPDC1 oncoantigen in malignant glioma and brain tumor initiating cells.

PubMed

Kikuchi, Ryogo; Sampetrean, Oltea; Saya, Hideyuki; Yoshida, Kazunari; Toda, Masahiro

2017-06-01

DEP domain containing 1 (DEPDC1) is a novel oncoantigen expressed in cancer cells, which presents oncogenic activity and high immunogenicity. Although DEPDC1 has been predicted to be a useful antigen for the development of a cancer vaccine, its pathophysiological roles in glioma have not been investigated. Here, we analyzed the expression and function of DEPDC1 in malignant glioma. DEPDC1 expression in glioma cell lines, glioma tissues, and brain tumor initiating cells (BTICs) was assessed by western blot and quantitative polymerase chain reaction (PCR). The effect of DEPDC1 downregulation on cell growth and nuclear factor kappa B (NFκB) signaling in glioma cells was investigated. Overall survival was assessed in mouse glioma models using human glioma cells and induced mouse brain tumor stem cells (imBTSCs) to determine the effect of DEPDC1 suppression in vivo. DEPDC1 expression was increased in glioma cell lines, tissues, and BTICs. Suppression of endogenous DEPDC1 expression by small interfering RNA (siRNA) inhibited glioma cell viability and induced apoptosis through NFκB signaling. In mouse glioma models using human glioma cells and imBTSCs, downregulation of DEPDC1 expression prolonged overall survival. These results suggest that DEPDC1 represents a target molecule for the treatment of glioma.

Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia1

PubMed Central

Pennycooke, Joyce C.; Jones, Michelle L.; Stushnoff, Cecil

2003-01-01

α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants. PMID:14500789

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells.

PubMed

Kirilyuk, Alexander; Tolstonog, Genrich V; Damert, Annette; Held, Ulrike; Hahn, Silvia; Löwer, Roswitha; Buschmann, Christian; Horn, Axel V; Traub, Peter; Schumann, Gerald G

2008-02-01

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

Type W Human Endogenous Retrovirus (HERV-W) Integrations and Their Mobilization by L1 Machinery: Contribution to the Human Transcriptome and Impact on the Host Physiopathology.

PubMed

Grandi, Nicole; Tramontano, Enzo

2017-06-27

Human Endogenous Retroviruses (HERVs) are ancient infection relics constituting ~8% of our DNA. While HERVs' genomic characterization is still ongoing, impressive amounts of data have been obtained regarding their general expression across tissues. Among HERVs, one of the most studied is the W group, which is the sole HERV group specifically mobilized by the long interspersed element-1 (LINE-1) machinery, providing a source of novel insertions by retrotransposition of HERV-W processed pseudogenes, and comprising a member encoding a functional envelope protein coopted for human placentation. The HERV-W group has been intensively investigated for its putative role in several diseases, such as cancer, inflammation, and autoimmunity. Despite major interest in the link between HERV-W expression and human pathogenesis, no conclusive correlation has been demonstrated so far. In general, (i) the absence of a proper identification of the specific HERV-W sequences expressed in a given condition, and (ii) the lack of studies attempting to connect the various observations in the same experimental conditions are the major problems preventing the definitive assessment of the HERV-W impact on human physiopathology. In this review, we summarize the current knowledge on the HERV-W group presence within the human genome and its expression in physiological tissues as well as in the main pathological contexts.

Type W Human Endogenous Retrovirus (HERV-W) Integrations and Their Mobilization by L1 Machinery: Contribution to the Human Transcriptome and Impact on the Host Physiopathology

PubMed Central

2017-01-01

Human Endogenous Retroviruses (HERVs) are ancient infection relics constituting ~8% of our DNA. While HERVs’ genomic characterization is still ongoing, impressive amounts of data have been obtained regarding their general expression across tissues. Among HERVs, one of the most studied is the W group, which is the sole HERV group specifically mobilized by the long interspersed element-1 (LINE-1) machinery, providing a source of novel insertions by retrotransposition of HERV-W processed pseudogenes, and comprising a member encoding a functional envelope protein coopted for human placentation. The HERV-W group has been intensively investigated for its putative role in several diseases, such as cancer, inflammation, and autoimmunity. Despite major interest in the link between HERV-W expression and human pathogenesis, no conclusive correlation has been demonstrated so far. In general, (i) the absence of a proper identification of the specific HERV-W sequences expressed in a given condition; and (ii) the lack of studies attempting to connect the various observations in the same experimental conditions are the major problems preventing the definitive assessment of the HERV-W impact on human physiopathology. In this review, we summarize the current knowledge on the HERV-W group presence within the human genome and its expression in physiological tissues as well as in the main pathological contexts. PMID:28653997

Serum amyloid A is an endogenous ligand that differentially induces IL-12 and IL-23.

PubMed

He, Rong; Shepard, Larry W; Chen, Jia; Pan, Zhixing K; Ye, Richard D

2006-09-15

The acute-phase proteins, C-reactive protein and serum amyloid A (SAA), are biomarkers of infection and inflammation. However, their precise role in immunity and inflammation remains undefined. We report in this study a novel property of SAA in the differential induction of Th1-type immunomodulatory cytokines IL-12 and IL-23. In peripheral blood monocytes and the THP-1 monocytic cell line, SAA induces the expression of IL-12p40, a subunit shared by IL-12 and IL-23. SAA-stimulated expression of IL-12p40 was rapid (< or = 4 h), sustainable (> or = 20 h), potent (up to 3380 pg/ml/10(6) cells in 24 h), and insensitive to polymyxin B treatment. The SAA-stimulated IL-12p40 secretion required de novo protein synthesis and was accompanied by activation of the transcription factors NF-kappaB and C/EBP. Expression of IL-12p40 required activation of the p38 MAPK and PI3K. Interestingly, the SAA-induced IL-12p40 production was accompanied by a sustained expression of IL-23p19, but not IL-12p35, resulting in preferential secretion of IL-23, but not IL-12. These results identify SAA as an endogenous ligand that potentially activates the IL-23/IL-17 pathway and present a novel mechanism for regulation of inflammation and immunity by an acute-phase protein.

A Focus on the Beneficial Effects of Alpha Synuclein and a Re-Appraisal of Synucleinopathies

PubMed Central

Ryskalin, Larisa; Busceti, Carla L.; Limanaqi, Fiona; Biagioni, Francesca; Gambardella, Stefano; Fornai, Francesco

2018-01-01

Alpha synuclein (α-syn) belongs to a class of proteins which are commonly considered to play a detrimental role in neuronal survival. This assumption is based on the occurrence of a severe neuronal degeneration in patients carrying a multiplication of the α-syn gene (SNCA) and in a variety of experi-mental models, where overexpression of α-syn leads to cell death and neurological impairment. In these conditions, a higher amount of normally structured α-syn produces a damage, which is even worse com-pared with that produced by α-syn owning an abnormal structure (as occurring following point gene muta-tions). In line with this, knocking out the expression of α-syn is reported to protect from specific neurotox-ins such as 1-methyl, 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the present review we briefly dis-cuss these well-known detrimental effects but we focus on findings showing that, in specific conditions α-syn is beneficial for cell survival. This occurs during methamphetamine intoxication which is counteracted by endogenous α-syn. Similarly, the dysfunction of the chaperone cysteine-string protein-alpha leads to cell pathology which is counteracted by over-expressing α-syn. In line with this, an increased expression of α-syn protects against oxidative damage produced by dopamine. Remarkably, when the lack of α-syn is combined with a depletion of β- and γ- synucleins, alterations in brain structure and function occur. This review tries to balance the evidence showing a beneficial effect with the bulk of data reporting a detri-mental effect of endogenous α-syn. The specific role of α-syn as a chaperone protein is discussed to ex-plain such a dual effect. PMID:29150919

A Focus on the Beneficial Effects of Alpha Synuclein and a Re-Appraisal of Synucleinopathies.

PubMed

Ryskalin, Larisa; Busceti, Carla L; Limanaqi, Fiona; Biagioni, Francesca; Gambardella, Stefano; Fornai, Francesco

2018-01-01

Alpha synuclein (α-syn) belongs to a class of proteins which are commonly considered to play a detrimental role in neuronal survival. This assumption is based on the occurrence of a severe neuronal degeneration in patients carrying a multiplication of the α-syn gene (SNCA) and in a variety of experimental models, where overexpression of α-syn leads to cell death and neurological impairment. In these conditions, a higher amount of normally structured α-syn produces a damage, which is even worse compared with that produced by α-syn owning an abnormal structure (as occurring following point gene mutations). In line with this, knocking out the expression of α-syn is reported to protect from specific neurotoxins such as 1-methyl, 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the present review we briefly discuss these well-known detrimental effects but we focus on findings showing that, in specific conditions α-syn is beneficial for cell survival. This occurs during methamphetamine intoxication which is counteracted by endogenous α-syn. Similarly, the dysfunction of the chaperone cysteine-string protein- alpha leads to cell pathology which is counteracted by over-expressing α-syn. In line with this, an increased expression of α-syn protects against oxidative damage produced by dopamine. Remarkably, when the lack of α-syn is combined with a depletion of β- and γ- synucleins, alterations in brain structure and function occur. This review tries to balance the evidence showing a beneficial effect with the bulk of data reporting a detrimental effect of endogenous α-syn. The specific role of α-syn as a chaperone protein is discussed to explain such a dual effect. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line.

PubMed

Chappell, Patrick E; White, Rachel S; Mellon, Pamela L

2003-12-03

Although it has long been established that episodic secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus is required for normal gonadotropin release, the molecular and cellular mechanisms underlying the synchronous release of GnRH are primarily unknown. We used the GT1-7 mouse hypothalamic cell line as a model for GnRH secretion, because these cells release GnRH in a pulsatile pattern similar to that observed in vivo. To explore possible molecular mechanisms governing secretory timing, we investigated the role of the molecular circadian clock in regulation of GnRH secretion. GT1-7 cells express many known core circadian clock genes, and we demonstrate that oscillations of these components can be induced by stimuli such as serum and the adenylyl cyclase activator forskolin, similar to effects observed in fibroblasts. Strikingly, perturbation of circadian clock function in GT1-7 cells by transient expression of the dominant-negative Clock-Delta19 gene disrupts normal ultradian patterns of GnRH secretion, significantly decreasing mean pulse frequency. Additionally, overexpression of the negative limb clock gene mCry1 in GT1-7 cells substantially increases GnRH pulse amplitude without a commensurate change in pulse frequency, demonstrating that an endogenous biological clock is coupled to the mechanism of neurosecretion in these cells and can regulate multiple secretory parameters. Finally, mice harboring a somatic mutation in the Clock gene are subfertile and exhibit a substantial increase in estrous cycle duration as revealed by examination of vaginal cytology. This effect persists in normal light/dark (LD) cycles, suggesting that a suprachiasmatic nucleus-independent endogenous clock in GnRH neurons is required for eliciting normal pulsatile patterns of GnRH secretion.

Riluzole activates TRPC5 channels independently of PLC activity

PubMed Central

Richter, Julia M; Schaefer, Michael; Hill, Kerstin

2014-01-01

BACKGROUND AND PURPOSE The transient receptor potential channel C5 (TRPC5) is a Ca2+-permeable cation channel, which is predominantly expressed in the brain. TRPC5 is activated in a PLC-dependent manner by, as yet, unidentified endogenous messengers. Recently, modulators of TRPC5, like Ca2+, pH and phospholipids, have been identified. However, the role of TRPC5 in vivo is only poorly understood. Novel specific modulators of TRPC5 might help to elucidate its function. EXPERIMENTAL APPROACH Novel modulators of TRPC5 were identified in a compound screening of approved drugs and natural compounds. The potency and selectivity of TRPC5-activating compounds were determined by fluorometric calcium imaging. The biophysical properties of channel activation by these compounds were analysed using electrophysiological measurements. KEY RESULTS Riluzole was identified as a novel activator of TRPC5 (EC50 9.2 ± 0.5 μM) and its mechanism of action was shown to be independent of G protein signalling and PLC activity. Riluzole-induced TRPC5 currents were potentiated by La3+ and, utilizing TRPC5 mutants that lack La3+ binding sites, it was confirmed that riluzole and La3+ activate TRPC5 by different mechanisms. Recordings of excised inside-out patches revealed a relatively direct effect of riluzole on TRPC5. CONCLUSIONS AND IMPLICATIONS Riluzole can activate TRPC5 heterologously expressed in HEK293 cells as well as those endogenously expressed in the U-87 glioblastoma cell line. Riluzole does not activate any other member of the TRPC family and could, therefore, despite its action on other ion channels, be a useful pharmacological tool for identifying TRPC5-specific currents in immortalized cell lines or in acutely isolated primary cells. PMID:24117252

The evolution and functional characterization of lined seahorse (Hippocampus erectus) CCKs involved in fasting and thermal stress response.

PubMed

Zhang, Huixian; Qin, Geng; Sun, Jinhui; Zhang, Bo; Lin, Qiang

2018-01-01

The peptide cholecystokinin (CCK) plays an important role in the regulation of vertebrate appetite and feeding behaviour. In the present study, the full-length cDNA and genomic DNA sequences of two CCK precursors were cloned and analysed in the Syngnathidae fish, the lined seahorse (Hippocampus erectus). Both CCK1 and CCK2 in the seahorse consist of four exons. The sequence of the octapeptide of seahorse CCK1 (DYMGWMDF) was the same as that of the chicken and human, while the octapeptide of seahorse CCK2 (DYEGWMDF) was unique among vertebrates. According to the phylogenetic analysis, two types of CCKs were produced by teleost-specific genome duplication (TGD). Both CCK1 and CCK2 were highly expressed in the brain, while detectable amounts of CCK1 mRNA in the brood pouch and CCK2 mRNA in the intestine were also found. Both CCK1 and CCK2 mRNA levels significantly increased during the transition from endogenous to exogenous nutrition. Additionally, fasting induced a significant increase in the CCK1 mRNA expression in the brain of juvenile seahorses but had no effect on CCK2 transcript levels. In addition, the CCK1 and CCK2 mRNA levels in the seahorse brain significantly increased after a high-temperature treatment. Thus, the mRNA expression of CCK had obvious tissue specificities and this preliminary study opens new avenues for further functional studies on the endocrine regulations of CCK in the transition from endogenous to exogenous nutrition, food intake regulation and metabolism in the seahorse. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation and characterization of Lhx9 – GFPCreERT2 knock-in mouse line

PubMed Central

Xie, Xiaoling; Deng, Min; Gan, Lin

2014-01-01

Summary LHX9 is a LIM-homeodomain transcription factor essential for the development of gonads, spinal cord interneurons, and thalamic neurons to name a few. We recently reported the expression of LHX9 in retinal amacrine cells during development. In this study, we generated an Lhx9 - GFPCreERT2 (GCE) knock-in mouse line by knocking-in a GCE cassette at the Lhx9 locus, thus inactivating endogenous Lhx9. Lhx9GCE/+ mice were viable, fertile, and displayed no overt phenotypical characteristics. Lhx9GCE/GCE mice were all phenotypically female, smaller in size, viable, but infertile. The specificity and efficacy of the Lhx9-GCE mouse line was verified by crossing it to a Rosa26 - tdTomato reporter mouse line, which reveals the Cre recombinase activities in retinal amacrine cells, developing limbs, testis, hippocampal neurons, thalamic neurons, and cerebellar neurons. Taken together, the Lhx9-GCE mouse line could serve as a beneficial tool for lineage tracing and gene manipulation experiments. PMID:25112520

Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

PubMed Central

Kregel, Steven; Kiriluk, Kyle J.; Rosen, Alex M.; Cai, Yi; Reyes, Edwin E.; Otto, Kristen B.; Tom, Westin; Paner, Gladell P.; Szmulewitz, Russell Z.; Vander Griend, Donald J.

2013-01-01

Despite advances in detection and therapy, castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways, and their regulation by the Androgen Receptor (AR), has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced, castrate-resistant prostate cancers. To this end, we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate, Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative, with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1, endogenous expression of Sox2 was repressed by AR signaling, and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise, in normal prostate epithelial cells and human embryonic stem cells, increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines, and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration, and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways. PMID:23326489

Promoter hypermethylation contributes to frequent inactivation of a putative conditional tumor suppressor gene connective tissue growth factor in ovarian cancer.

PubMed

Kikuchi, Ryoko; Tsuda, Hitoshi; Kanai, Yae; Kasamatsu, Takahiro; Sengoku, Kazuo; Hirohashi, Setsuo; Inazawa, Johji; Imoto, Issei

2007-08-01

Connective tissue growth factor (CTGF) is a secreted protein belonging to the CCN family, members of which are implicated in various biological processes. We identified a homozygous loss of CTGF (6q23.2) in the course of screening a panel of ovarian cancer cell lines for genomic copy number aberrations using in-house array-based comparative genomic hybridization. CTGF mRNA expression was observed in normal ovarian tissue and immortalized ovarian epithelial cells but was reduced in many ovarian cancer cell lines without its homozygous deletion (12 of 23 lines) and restored after treatment with 5-aza 2'-deoxycytidine. The methylation status around the CTGF CpG island correlated inversely with the expression, and a putative target region for methylation showed promoter activity. CTGF methylation was frequently observed in primary ovarian cancer tissues (39 of 66, 59%) and inversely correlated with CTGF mRNA expression. In an immunohistochemical analysis of primary ovarian cancers, CTGF protein expression was frequently reduced (84 of 103 cases, 82%). Ovarian cancer tended to lack CTGF expression more frequently in the earlier stages (stages I and II) than the advanced stages (stages III and IV). CTGF protein was also differentially expressed among histologic subtypes. Exogenous restoration of CTGF expression or treatment with recombinant CTGF inhibited the growth of ovarian cancer cells lacking its expression, whereas knockdown of endogenous CTGF accelerated growth of ovarian cancer cells with expression of this gene. These results suggest that epigenetic silencing by hypermethylation of the CTGF promoter leads to a loss of CTGF function, which may be a factor in the carcinogenesis of ovarian cancer in a stage-dependent and/or histologic subtype-dependent manner.

A meta-analysis to evaluate the cellular processes regulated by the interactome of endogenous and over-expressed estrogen receptor alpha.

PubMed

Simões, Joana; Amado, Francisco M; Vitorino, Rui; Helguero, Luisa A

2015-01-01

The nature of the proteins complexes that regulate ERα subcellular localization and activity is still an open question in breast cancer biology. Identification of such complexes will help understand development of endocrine resistance in ER+ breast cancer. Mass spectrometry (MS) has allowed comprehensive analysis of the ERα interactome. We have compared six published works analyzing the ERα interactome of MCF-7 and HeLa cells in order to identify a shared or different pathway-related fingerprint. Overall, 806 ERα interacting proteins were identified. The cellular processes were differentially represented according to the ERα purification methodology, indicating that the methodologies used are complementary. While in MCF-7 cells, the interactome of endogenous and over-expressed ERα essentially represents the same biological processes and cellular components, the proteins identified were not over-lapping; thus, suggesting that the biological response may differ as the regulatory/participating proteins in these complexes are different. Interestingly, biological processes uniquely associated to ERα over-expressed in HeLa cell line included L-serine biosynthetic process, cellular amino acid biosynthetic process and cell redox homeostasis. In summary, all the approaches analyzed in this meta-analysis are valid and complementary; in particular, for those cases where the processes occur at low frequency with normal ERα levels, and can be identified when the receptor is over-expressed. However special effort should be put into validating these findings in cells expressing physiological ERα levels.

Functional Characterization of PaLAX1, a Putative Auxin Permease, in Heterologous Plant Systems1[W][OA

PubMed Central

Hoyerová, Klára; Perry, Lucie; Hand, Paul; Laňková, Martina; Kocábek, Tomáš; May, Sean; Kottová, Jana; Pačes, Jan; Napier, Richard; Zažímalová, Eva

2008-01-01

We have isolated the cDNA of the gene PaLAX1 from a wild cherry tree (Prunus avium). The gene and its product are highly similar in sequences to both the cDNAs and the corresponding protein products of AUX/LAX-type genes, coding for putative auxin influx carriers. We have prepared and characterized transformed Nicotiana tabacum and Arabidopsis thaliana plants carrying the gene PaLAX1. We have proved that constitutive overexpression of PaLAX1 is accompanied by changes in the content and distribution of free indole-3-acetic acid, the major endogenous auxin. The increase in free indole-3-acetic acid content in transgenic plants resulted in various phenotype changes, typical for the auxin-overproducing plants. The uptake of synthetic auxin, 2,4-dichlorophenoxyacetic acid, was 3 times higher in transgenic lines compared to the wild-type lines and the treatment with the auxin uptake inhibitor 1-naphthoxyacetic acid reverted the changes caused by the expression of PaLAX1. Moreover, the agravitropic response could be restored by expression of PaLAX1 in the mutant aux1 plants, which are deficient in auxin influx carrier activity. Based on our data, we have concluded that the product of the gene PaLAX1 promotes the uptake of auxin into cells, and, as a putative auxin influx carrier, it affects the content and distribution of free endogenous auxin in transgenic plants. PMID:18184737

RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells.

PubMed

Han, Shujie; Knoepp, Stewart M; Hallman, Mark A; Meier, Kathryn E

2007-01-01

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk mitogen-activated protein kinase activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells but not in resistant cells lacking Ras-GRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of small interfering RNA (siRNA) for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA-sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

Notch3 signaling-mediated melanoma-endothelial crosstalk regulates melanoma stem-like cell homeostasis and niche morphogenesis.

PubMed

Hsu, Mei-Yu; Yang, Moon Hee; Schnegg, Caroline I; Hwang, Soonyean; Ryu, Byungwoo; Alani, Rhoda M

2017-06-01

Melanoma is among the most virulent cancers, owing to its propensity to metastasize and its resistance to current therapies. The treatment failure is largely attributed to tumor heterogeneity, particularly subpopulations possessing stem cell-like properties, ie, melanoma stem-like cells (MSLCs). Evidence indicates that the MSLC phenotype is malleable and may be acquired by non-MSLCs through phenotypic switching upon appropriate stimuli, the so-called 'dynamic stemness'. Since the phenotypic characteristics and functional integrity of MSLCs depend on their vascular niche, using a two-dimensional (2D) melanoma-endothelium co-culture model, where the MSLC niche is recapitulated in vitro, we identified Notch3 signaling pathway as a micro-environmental cue governing MSLC phenotypic plasticity via pathway-specific gene expression arrays. Accordingly, lentiviral shRNA-mediated Notch3 knockdown (KD) in melanoma cell lines exhibiting high levels of endogenous Notch3 led to retarded/abolished tumorigenicity in vivo through both depleting MSLC fractions, evinced by MSLC marker downregulation (eg, CD133 and CD271); and impeding the MSLC niche, corroborated by the attenuated tumor angiogenesis as well as vasculogenic mimicry. In contrast, Notch3 KD affected neither tumor growth nor MSLC subsets in a melanoma cell line with relatively low endogenous Notch3 expression. Thus, Notch3 signaling may facilitate MSLC plasticity and niche morphogenesis in a cell context-dependent manner. Our findings illustrate Notch3 as a molecular switch driving melanoma heterogeneity, and provide the biological rationale for Notch inhibition as a promising therapeutic option.

Regulation of retinoic acid synthetic enzymes by WT1 and HDAC inhibitors in 293 cells.

PubMed

Li, Yifan; Wang, Lei; Ai, Weipeng; He, Nianhui; Zhang, Lin; Du, Jihui; Wang, Yong; Mao, Xingjian; Ren, Junqi; Xu, Dan; Zhou, Bei; Li, Rong; Mai, Liwen

2017-09-01

All-trans retinoic acid (atRA), which is mainly generated endogenously via two steps of oxidation from vitamin A (retinol), plays an indispensible role in the development of the kidney and many other organs. Enzymes that catalyze the oxidation of retinol to generate atRA, including aldehyde dehydrogenase 1 family (ALDH1)A1, ALDH1A2 and ALDH1A3, exhibit complex expression patterns at different stages of renal development. However, molecular triggers that control these differential expression levels are poorly understood. In this study, we provide in vitro evidence to demonstrate that Wilms' tumor 1 (WT1) negatively regulates the expression of the atRA synthetic enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, in the 293 cell line, leading to significant blockage of atRA production. Furthermore, we demonstrate that the suppression of ALDH1A1 by WT1 can be markedly attenuated by histone deacetylase inhibitors (HDACis). Taken together, we provide evidence to indicate that WT1 and HDACs are strong regulators of endogenous retinoic acid synthetic enzymes in 293 cells, indicating that they may be involved in the regulation of atRA synthesis.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.

PubMed

Guo, Jianying; Ma, Dacheng; Huang, Rujin; Ming, Jia; Ye, Min; Kee, Kehkooi; Xie, Zhen; Na, Jie

2017-05-01

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

PubMed

Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

2011-09-01

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Immunohistochemical Analysis Using Antipodocalyxin Monoclonal Antibody PcMab-47 Demonstrates Podocalyxin Expression in Oral Squamous Cell Carcinomas.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan, and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed in many types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized mice with recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced a novel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxin-expressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry, and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern and detected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.

Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP2 in dorsal root ganglia neurons.

PubMed

Coutinho-Budd, Jaeda C; Snider, Samuel B; Fitzpatrick, Brendan J; Rittiner, Joseph E; Zylka, Mark J

2013-09-08

Rapamycin-induced translocation systems can be used to manipulate biological processes with precise temporal control. These systems are based on rapamycin-induced dimerization of FK506 Binding Protein 12 (FKBP12) with the FKBP Rapamycin Binding (FRB) domain of mammalian target of rapamycin (mTOR). Here, we sought to adapt a rapamycin-inducible phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphatase (Inp54p) system to deplete PIP2 in nociceptive dorsal root ganglia (DRG) neurons. We genetically targeted membrane-tethered CFP-FRBPLF (a destabilized FRB mutant) to the ubiquitously expressed Rosa26 locus, generating a Rosa26-FRBPLF knockin mouse. In a second knockin mouse line, we targeted Venus-FKBP12-Inp54p to the Calcitonin gene-related peptide-alpha (CGRPα) locus. We hypothesized that after intercrossing these mice, rapamycin treatment would induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in CGRP+ DRG neurons. In control experiments with cell lines, rapamycin induced translocation of Venus-FKBP12-Inp54p to the plasma membrane, and subsequent depletion of PIP2, as measured with a PIP2 biosensor. However, rapamycin did not induce translocation of Venus-FKBP12-Inp54p to the plasma membrane in FRBPLF-expressing DRG neurons (in vitro or in vivo). Moreover, rapamycin treatment did not alter PIP2-dependent thermosensation in vivo. Instead, rapamycin treatment stabilized FRBPLF in cultured DRG neurons, suggesting that rapamycin promoted dimerization of FRBPLF with endogenous FKBP12. Taken together, our data indicate that these knockin mice cannot be used to inducibly deplete PIP2 in DRG neurons. Moreover, our data suggest that high levels of endogenous FKBP12 could compete for binding to FRBPLF, hence limiting the use of rapamycin-inducible systems to cells with low levels of endogenous FKBP12.

Expression of Ca2+-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells

PubMed Central

Ruas, Margarida; Davis, Lianne C; Chen, Cheng-Chang; Morgan, Anthony J; Chuang, Kai-Ting; Walseth, Timothy F; Grimm, Christian; Garnham, Clive; Powell, Trevor; Platt, Nick; Platt, Frances M; Biel, Martin; Wahl-Schott, Christian; Parrington, John; Galione, Antony

2015-01-01

The second messenger NAADP triggers Ca2+ release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2−/−), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca2+ responses as assessed by single-cell Ca2+ imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2−/− cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca2+-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca2+ release. High-affinity [32P]NAADP binding still occurs in Tpcn1/2−/− tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca2+-permeable channels indispensable for NAADP signalling. PMID:25872774

MCT1 promotes the cisplatin-resistance by antagonizing Fas in epithelial ovarian cancer.

PubMed

Yan, Chunxiao; Yang, Fan; Zhou, Chunxia; Chen, Xuejun; Han, Xuechuan; Liu, Xueqin; Ma, Hongyun; Zheng, Wei

2015-01-01

This study was designed to investigate the role of MCT1 in the development of cisplatin-resistant ovarian cancer and its possible relationship with Fas. We found the expression of MCT1 was obviously increased both in cisplatin-resistant ovarian cancer tissue and A2780/CP cells compared with sensitive ovarian cancer tissue and cell lines A2780. And in A2780 cells treated with Cisplatin, the expression of MCT1 increased in a concentration-dependent manner, MCT1 knockdown attenuates cisplatin-induced cell viability. In A2780 and A2780/CP cells transfected with MCT1 siRNA, the activation of several downstream targets of Fas, including FasL and FAP-1 were largely prevented, whereas the expression of Caspase-3 was increased, accompanying with increased abundance of Fas. Coimmunoprecipitation and immunofluorescence showed that there is interaction between endogenous MCT1 with Fas in vivo and in vitro. In vivo, depletion of MCT1 by shRNA reverses cisplatin-resistance and the expression of Fas. This study showed that down regulation of MCT1 promote the sensibility to Cisplatin in ovarian cancer cell line. And this effect appeared to be mediated via antagonizing the effect of Fas.

MCT1 promotes the cisplatin-resistance by antagonizing Fas in epithelial ovarian cancer

PubMed Central

Yan, Chunxiao; Yang, Fan; Zhou, Chunxia; Chen, Xuejun; Han, Xuechuan; Liu, Xueqin; Ma, Hongyun; Zheng, Wei

2015-01-01

This study was designed to investigate the role of MCT1 in the development of cisplatin-resistant ovarian cancer and its possible relationship with Fas. We found the expression of MCT1 was obviously increased both in cisplatin-resistant ovarian cancer tissue and A2780/CP cells compared with sensitive ovarian cancer tissue and cell lines A2780. And in A2780 cells treated with Cisplatin, the expression of MCT1 increased in a concentration-dependent manner, MCT1 knockdown attenuates cisplatin-induced cell viability. In A2780 and A2780/CP cells transfected with MCT1 siRNA, the activation of several downstream targets of Fas, including FasL and FAP-1 were largely prevented, whereas the expression of Caspase-3 was increased, accompanying with increased abundance of Fas. Coimmunoprecipitation and immunofluorescence showed that there is interaction between endogenous MCT1 with Fas in vivo and in vitro. In vivo, depletion of MCT1 by shRNA reverses cisplatin-resistance and the expression of Fas. This study showed that down regulation of MCT1 promote the sensibility to Cisplatin in ovarian cancer cell line. And this effect appeared to be mediated via antagonizing the effect of Fas. PMID:26045776

Multiple effects of genetic background on variegated transgene expression in mice.

PubMed Central

Opsahl, Margaret L; McClenaghan, Margaret; Springbett, Anthea; Reid, Sarah; Lathe, Richard; Colman, Alan; Whitelaw, C Bruce A

2002-01-01

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression. PMID:11901126

Multiple effects of genetic background on variegated transgene expression in mice.

PubMed

Opsahl, Margaret L; McClenaghan, Margaret; Springbett, Anthea; Reid, Sarah; Lathe, Richard; Colman, Alan; Whitelaw, C Bruce A

2002-03-01

BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death.

PubMed

Almeida, Ana S; Soares, Nuno L; Vieira, Melissa; Gramsbergen, Jan Bert; Vieira, Helena L A

2016-01-01

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO's improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO's increasing number of differentiated neurons in OHSC. In conclusion, CO's increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO's effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies.

Carbon Monoxide Releasing Molecule-A1 (CORM-A1) Improves Neurogenesis: Increase of Neuronal Differentiation Yield by Preventing Cell Death

PubMed Central

Almeida, Ana S.; Soares, Nuno L.; Vieira, Melissa; Gramsbergen, Jan Bert

2016-01-01

Cerebral ischemia and neurodegenerative diseases lead to impairment or death of neurons in the central nervous system. Stem cell based therapies are promising strategies currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase (HO) activity. Administration of CO at low concentrations produces several beneficial effects in distinct tissues, namely anti-apoptotic and anti-inflammatory. Herein the CO role on modulation of neuronal differentiation was assessed. Three different models with increasing complexity were used: human neuroblastoma SH-S5Y5 cell line, human teratocarcinoma NT2 cell line and organotypic hippocampal slice cultures (OHSC). Cell lines were differentiated into post-mitotic neurons by treatment with retinoic acid (RA) supplemented with CO-releasing molecule A1 (CORM-A1). CORM-A1 positively modulated neuronal differentiation, since it increased final neuronal production and enhanced the expression of specific neuronal genes: Nestin, Tuj1 and MAP2. Furthermore, during neuronal differentiation process, there was an increase in proliferative cell number (ki67 mRNA expressing cells) and a decrease in cell death (lower propidium iodide (PI) uptake, limitation of caspase-3 activation and higher Bcl-2 expressing cells). CO supplementation did not increase the expression of RA receptors. In the case of SH-S5Y5 model, small amounts of reactive oxygen species (ROS) generation emerges as important signaling molecules during CO-promoted neuronal differentiation. CO’s improvement of neuronal differentiation yield was validated using OHSC as ex vivo model. CORM-A1 treatment of OHSC promoted higher levels of cells expressing the neuronal marker Tuj1. Still, CORM-A1 increased cell proliferation assessed by ki67 expression and also prevented cell death, which was followed by increased Bcl-2 expression, decreased levels of active caspase-3 and PI uptake. Likewise, ROS signaling emerged as key factors in CO’s increasing number of differentiated neurons in OHSC. In conclusion, CO’s increasing number of differentiated neurons is a novel biological role disclosed herein. CO improves neuronal yield due to its capacity to reduce cell death, promoting an increase in proliferative population. However, one cannot disregard a direct CO’s effect on specific cellular processes of neuronal differentiation. Further studies are needed to evaluate how CO can potentially modulate cell mechanisms involved in neuronal differentiation. In summary, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies. PMID:27144388

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs.

PubMed

Khan, Aly A; Betel, Doron; Miller, Martin L; Sander, Chris; Leslie, Christina S; Marks, Debora S

2009-06-01

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation.

PubMed

Cui, Yan; Ying, Ying; van Hasselt, Andrew; Ng, Ka Man; Yu, Jun; Zhang, Qian; Jin, Jie; Liu, Dingxie; Rhim, Johng S; Rha, Sun Young; Loyo, Myriam; Chan, Anthony T C; Srivastava, Gopesh; Tsao, George S W; Sellar, Grant C; Sung, Joseph J Y; Sidransky, David; Tao, Qian

2008-08-20

Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction. Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing. Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.

Retrotransposon expression and incorporation of cloned human and mouse retroelements in human spermatozoa.

PubMed

Lazaros, Leandros; Kitsou, Chrysoula; Kostoulas, Charilaos; Bellou, Sofia; Hatzi, Elissavet; Ladias, Paris; Stefos, Theodoros; Markoula, Sofia; Galani, Vasiliki; Vartholomatos, Georgios; Tzavaras, Theodore; Georgiou, Ioannis

2017-03-01

To investigate the expression of long interspersed element (LINE) 1, human endogenous retrovirus (HERV) K10, and short interspersed element-VNTR-Alu element (SVA) retrotransposons in ejaculated human spermatozoa by means of reverse-transcription (RT) polymerase chain reaction (PCR) analysis as well as the potential incorporation of cloned human and mouse active retroelements in human sperm cell genome. Laboratory study. University research laboratories and academic hospital. Normozoospermic and oligozoospermic white men. RT-PCR analysis was performed to confirm the retrotransposon expression in human spermatozoa. Exogenous retroelements were tagged with a plasmid containing a green fluorescence (EGFP) retrotransposition cassette, and the de novo retrotransposition events were tested with the use of PCR, fluorescence-activated cell sorting analysis, and confocal microscopy. Retroelement expression in human spermatozoa, incorporation of cloned human and mouse active retroelements in human sperm genome, and de novo retrotransposition events in human spermatozoa. RT-PCR products of expressed human LINE-1, HERV-K10, and SVA retrotransposons were observed in ejaculated human sperm samples. The incubation of human spermatozoa with either retrotransposition-active human LINE-1 and HERV-K10 or mouse reverse transcriptase-deficient VL30 retrotransposons tagged with an EGFP-based retrotransposition cassette led to EGFP-positive spermatozo; 16.67% of the samples were positive for retrotransposition. The respective retrotransposition frequencies for the LINE-1, HERV-K10, and VL30 retrotransposons in the positive samples were 0.34 ± 0.13%, 0.37 ± 0.17%, and 0.30 ± 0.14% per sample of 10,000 spermatozoa. Our results show that: 1) LINE-1, HERV-K10, and SVA retrotransposons are transcriptionally expressed in human spermatozoa; 2) cloned active retroelements of human and mammalian origin can be incorporated in human sperm genome; 3) active reverse transcriptases exist in human spermatozoa; and 4) de novo retrotransposition events occur in human spermatozoa. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

A Jurkat 76 based triple parameter reporter system to evaluate TCR functions and adoptive T cell strategies.

PubMed

Rosskopf, Sandra; Leitner, Judith; Paster, Wolfgang; Morton, Laura T; Hagedoorn, Renate S; Steinberger, Peter; Heemskerk, Mirjam H M

2018-04-03

Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.

Meeting Report: The Role of the Mobilome in Cancer

PubMed Central

Ardeljan, Daniel; Taylor, Martin S.; Burns, Kathleen H.; Boeke, Jef D.; Espey, Michael Graham; Woodhouse, Elisa C.; Howcroft, T. Kevin

2016-01-01

Approximately half of the human genome consists of repetitive sequence attributed to the activities of mobile DNAs, including DNA transposons, RNA transposons, and endogenous retroviruses. Of these, only Long INterspersed Elements (LINE-1 or L1) and sequences copied by LINE-1 remain mobile in our species today. Although cells restrict L1 activity by both transcriptional and post-transcriptional mechanisms, L1 de-repression occurs in developmental and pathologic contexts, including many types of cancers. However, we have limited knowledge of the extent and consequences of L1 expression in premalignancies and cancer. Participants in this NIH strategic workshop considered key questions to enhance our understanding of mechanisms and roles the mobilome may play in cancer biology. PMID:27527733

Inhibition of connective tissue growth factor (CTGF/CCN2) in gallbladder cancer cells leads to decreased growth in vitro.

PubMed

Garcia, Patricia; Leal, Pamela; Ili, Carmen; Brebi, Priscilla; Alvarez, Hector; Roa, Juan C

2013-06-01

Gallbladder cancer (GBC) is an aggressive neoplasm associated with late diagnosis, unsatisfactory treatment and poor prognosis. Previous work showed that connective tissue growth factor (CTGF) expression is increased in this malignancy. This matricellular protein plays an important role in various cellular processes and its involvement in the tumorigenesis of several human cancers has been demonstrated. However, the precise function of CTGF expression in cancer cells is yet to be determined. The aim of this study was to evaluate the CTGF expression in gallbladder cancer cell lines, and its effect on cell viability, colony formation and in vitro cell migration. CTGF expression was evaluated in seven GBC cell lines by Western blot assay. Endogenous CTGF expression was downregulated by lentiviral shRNA directed against CTGF mRNA in G-415 cells, and the effects on cell viability, anchorage-independent growth and migration was assessed by comparing them to scrambled vector-transfected cells. Knockdown of CTGF resulted in significant reduction in cell viability, colony formation and anchorage-independent growth (P < 0.05). An increased p27 expression was observed in G-415 cells with loss of CTGF function. Our results suggest that high expression of this protein in gallbladder cancer may confer a growth advantage for neoplastic cells. © 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.

Efficacy of ATR inhibitors as single agents in Ewing sarcoma

PubMed Central

Lecona, Emilio; Murga, Matilde; Callen, Elsa; Azorin, Daniel; Alonso, Javier; Lopez, Andres J.; Nussenzweig, Andre; Fernandez-Capetillo, Oscar

2016-01-01

Ewing sarcomas (ES) are pediatric bone tumors that arise from a driver translocation, most frequently EWS/FLI1. Current ES treatment involves DNA damaging agents, yet the basis for the sensitivity to these therapies remains unknown. Oncogene-induced replication stress (RS) is a known source of endogenous DNA damage in cancer, which is suppressed by ATR and CHK1 kinases. We here show that ES suffer from high endogenous levels of RS, rendering them particularly dependent on the ATR pathway. Accordingly, two independent ATR inhibitors show in vitro toxicity in ES cell lines as well as in vivo efficacy in ES xenografts as single agents. Expression of EWS/FLI1 or EWS/ERG oncogenic translocations sensitizes non-ES cells to ATR inhibitors. Our data shed light onto the sensitivity of ES to genotoxic agents, and identify ATR inhibitors as a potential therapy for Ewing Sarcomas. PMID:27577084

Methionine sulfoximine supplementation enhances productivity in GS-CHOK1SV cell lines through glutathione biosynthesis.

PubMed

Feary, Marc; Racher, Andrew J; Young, Robert J; Smales, C Mark

2017-01-01

In Lonza Biologics' GS Gene Expression System™, recombinant protein-producing GS-CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine-free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER-resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS-CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS-CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb-producing GS-CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight-fold and the concentration in harvest medium by two-fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process-driven method for increasing mAb productivity from GS-CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17-25, 2017. © 2016 American Institute of Chemical Engineers.

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

PubMed

Merkl, Claudia; Saalfrank, Anja; Riesen, Nathalie; Kühn, Ralf; Pertek, Anna; Eser, Stefan; Hardt, Markus Sebastian; Kind, Alexander; Saur, Dieter; Wurst, Wolfgang; Iglesias, Antonio; Schnieke, Angelika

2013-01-01

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

PubMed

Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

1996-08-01

To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

Effects of Spaceflight on Drosophila Neural Development

NASA Technical Reports Server (NTRS)

Keshishian, Haig S.

1997-01-01

The major goal from the animal side, however, has been achieved, namely to develop Drosophila lines where we can assay individual neuromuscular endings directly without dissection. This was achieved by means of using the GAL4-UAS system, where we have succeeded in establishing stocks of flies where the key neuromuscular connections can be assayed directly in undissected larvae by means of the expression of endogenously fluorescent reporters in the specific motor endings. The green fluorescent protein (GFP) as a reporter allows scoring of neural anatomy en-masse in whole mount using fluorescent microscopy without the need for either dissection or specific labeling. Two stocks have been developed. The first, which we developed first, uses the S65T mutant form, which has a dramatically brighter expression than the native protein. This animal will use GAL4 drivers with expression under the control of the elav gene, and which will ensure expression in all neurons of the embryo and larva. The second transgenic animal we have developed is of a novel kind, and makes use of dicistronic design, so that two copies of the protein will be expressed per insert. We have also developed a tricistronic form, but this has not yet been transformed into flies, and we do not imagine that this third line will be ready in time for the flight.

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

PubMed Central

Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

Long non-coding RNA MIAT promotes breast cancer progression and functions as ceRNA to regulate DUSP7 expression by sponging miR-155-5p.

PubMed

Luan, Tian; Zhang, Ximei; Wang, Shuyuan; Song, Yan; Zhou, Shunheng; Lin, Jing; An, Weiwei; Yuan, Weiguang; Yang, Yue; Cai, Huilong; Zhang, Qingyuan; Wang, Lihong

2017-09-29

Long non-coding RNAs (lncRNA) have been reported as key regulators in the progression and metastasis of breast cancer. In this study, we found that the lncRNA myocardial infarction associated transcript (MIAT) expression was upregulated in breast cancer in The Cancer Genome Atlas (TCGA) data sets. We validated that MIAT was higher in breast cancer cell lines and advanced breast tumors than in normal controls. And MIAT overexpression associated with TNM stage and lymphnode metastasis. Knockdown MIAT inhibited breast cancer cell proliferation and promoted apoptosis. Also MIAT downregulation suppressed epithelial-mesenchymal transition (EMT) and decreased migration and invasion in MDA-MB-231 and MCF-7 breast cancer cell lines. More importantly, knockdown MIAT inhibited tumor growth in vivo . Our results suggested that MIAT acted as a competing endogenous RNA (ceRNA) to regulate the expression of dual specificity phosphatase 7 (DUSP7) by taking up miR-155-5p in breast cancer. There were positive correlation between MIAT and DUSP7 expression in breast cancer patients. We conclude that MIAT promotes breast cancer progression and functions as ceRNA to regulate DUSP7 expression by sponging miR-155-5p in breast cancer.

miR-297 modulates multidrug resistance in human colorectal carcinoma by down-regulating MRP-2.

PubMed

Xu, Ke; Liang, Xin; Shen, Ke; Cui, Daling; Zheng, Yuanhong; Xu, Jianhua; Fan, Zhongze; Qiu, Yanyan; Li, Qi; Ni, Lei; Liu, Jianwen

2012-09-01

Colorectal carcinoma is a frequent cause of cancer-related death in men and women. miRNAs (microRNAs) are endogenous small non-coding RNAs that regulate gene expression negatively at the post-transcriptional level. In the present study we investigated the possible role of microRNAs in the development of MDR (multidrug resistance) in colorectal carcinoma cells. We analysed miRNA expression levels between MDR colorectal carcinoma cell line HCT116/L-OHP cells and their parent cell line HCT116 using a miRNA microarray. miR-297 showed lower expression in HCT116/L-OHP cells compared with its parental cells. MRP-2 (MDR-associated protein 2) is an important MDR protein in platinum-drug-resistance cells and is a predicted target of miR-297. Additionally miR-297 was down-regulated in a panel of human colorectal carcinoma tissues and negatively correlated with expression levels of MRP-2. Furthermore, we found that ectopic expression of miR-297 in MDR colorectal carcinoma cells reduced MRP-2 protein level and sensitized these cells to anti-cancer drugs in vitro and in vivo. Taken together, our findings suggest that miR-297 could play a role in the development of MDR in colorectal carcinoma cells, at least in part by modulation of MRP-2.

An endogenous 55 kDa TNF receptor mediates cell death in a neural cell line.

PubMed

Sipe, K J; Srisawasdi, D; Dantzer, R; Kelley, K W; Weyhenmeyer, J A

1996-06-01

Tumor necrosis factor-alpha (TNF) is associated with developmental and injury-related events in the central nervous system (CNS). In the present study, we have examined the role of TNF on neurons using the clonal murine neuroblastoma line, N1E-115 (N1E). N1E cells represent a well-defined model for studying neuronal development since they can be maintained as either undifferentiated, mitotically active neuroblasts or as differentiated, mature neurons. Northern and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that both undifferentiated and differentiated N1Es express transcripts for the 55 kDa TNF receptor (TNFR), but not the 75 kDa TNFR. The biological activity of the expressed TNF receptor was demonstrated by a dose dependent cytotoxicity to either recombinant murine or human TNF when the cells were incubated with the transcriptional inhibitor actinomycin D. The lack of the 75 kDa receptor mRNA expression and the dose dependent response to rHuTNF, an agonist specific for the murine 55 kDa receptor, suggest that the TNF induced cytotoxicity is mediated through the 55 kDa receptor in both the undifferentiated and differentiated N1Es. Light microscopic observations, flow cytometric analysis of hypodiploid DNA, and electrophoretic analysis of nucleosomal DNA fragmentation of N1Es treated with actinomycin D and TNF revealed features characteristic of both necrotic and apoptotic cell death. These findings demonstrate that blast and mature N1E cells express the 55 kDa TNF receptor which is responsible for inducing both necrotic and apoptotic death in these cells. The observation that actinomycin D renders N1E cells susceptible to the cytotoxic effects of TNF indicates that a sensitization step, such as removal of an endogenous protective factor or viral-mediated inhibition of transcription, may be necessary for TNF cytotoxicity in neurons.

Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

PubMed

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

PubMed Central

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

Herpes simplex virus regulatory proteins VP16 and ICP0 counteract an innate intranuclear barrier to viral gene expression.

PubMed

Hancock, Meaghan H; Corcoran, Jennifer A; Smiley, James R

2006-08-15

HSV regulatory proteins VP16 and ICP0 play key roles in launching the lytic program of viral gene expression in most cell types. However, these activation functions are dispensable in U2OS osteosarcoma cells, suggesting that this cell line either expresses an endogenous activator of HSV gene expression or lacks inhibitory mechanisms that are inactivated by VP16 and ICP0 in other cells. To distinguish between these possibilities, we examined the phenotypes of somatic cell hybrids formed between U2OS cells and highly restrictive HEL fibroblasts. The U2OS-HEL heterokarya were as non-permissive as HEL cells, a phenotype that could be overcome by providing either VP16 or ICP0 in trans. Our data indicate that human fibroblasts contain one or more inhibitory factors that act within the nucleus to limit HSV gene expression and argue that VP16 and ICP0 stimulate viral gene expression at least in part by counteracting this innate antiviral defence mechanism.

Expression pattern of the pre-prothaumatin II gene under the control of the CaMV 35S promoter in transgenic cucumber (Cucumis sativus L.) flower buds and fruits.

PubMed

Szwacka, M; Siedlecka, E; Zawirska-Wojtasiak, R; Wiśniewski, Ł; Malepszy, S

2009-01-01

Thaumatin II is an extremely sweet-tasting protein produced by fruits of the West African shrub Thaumatococcus daniellii Benth, so it can be used in biotechnology to improve the tastes of various plant products. This study is concerned with the spatial and temporal aspects of expression of the 35S-pre-prothaumatin II chimeric gene in flower buds and fruits of transgenic cucumber (Cucumis sativus L.) line 225. The activity of the 35S promoter in organs of line 225 was compared with its activity in 2 other transgenic lines. The accumulation of recombinant thaumatin varied spatially in flower bud tissues of transgenic lines. We found that these differences in the spatial accumulation of transgenic protein concerned the ovary of female buds and the perianth of male buds. In contrast to flower parts, recombinant thaumatin was found in nearly all parts of the young fruit from the transgenic plants. The pre-prothaumatin II gene expression was detected at a very early developmental stage in male buds, and its pattern was rather conserved as the buds aged. The expression of the transgene was also detected in vascular tissues of examined organs but was undetectable in pollen grains, in agreement with the generally held view that the CaMV 35S promoter is virtually silent in pollen. Immunocytochemical analyses of sections of control organs revealed endogenous homolog(s) of thaumatin when using polyclonal antisera, but not when using monoclonal antibodies for recombinant thaumatin detection in transgenic cucumber.

Low Incidence along with Low mRNA Levels of EGFRvIII in Prostate and Colorectal Cancers Compared to Glioblastoma

PubMed Central

Peciak, Joanna; Stec, Wojciech J; Treda, Cezary; Ksiazkiewicz, Magdalena; Janik, Karolina; Popeda, Marta; Smolarz, Maciej; Rosiak, Kamila; Hulas-Bigoszewska, Krystyna; Och, Waldemar; Rieske, Piotr; Stoczynska-Fidelus, Ewelina

2017-01-01

Background: The presence as well as the potential role of EGFRvIII in tumors other than glioblastoma still remains a controversial subject with many contradictory data published. Previous analyses, however, did not consider the level of EGFRvIII mRNA expression in different tumor types. Methods: Appropriately designed protocol for Real-time quantitative reverse-transcription PCR (Real-time qRT-PCR) was applied to analyze EGFRvIII and EGFRWT mRNA expression in 155 tumor specimens. Additionally, Western Blot (WB) analysis was performed for selected samples. Stable cell lines showing EGFRvIII expression (CAS-1 and DK-MG) were analyzed by means of WB, immunocytochemistry (ICC) and fluorescence in situ hybridization (FISH). Results: Our analyses revealed EGFRvIII expression in 27.59% of glioblastomas (8/29), 8.11% of colorectal cancers (3/37), 6.52% of prostate cancers (3/46) and none of breast cancers (0/43). Despite the average relative expression of EGFRvIII varying greatly among tumors of different tissues (approximately 800-fold) or even within the same tissue group (up to 8000-fold for GB), even the marginal expression of EGFRvIII mRNA can be detrimental to cancer progression, as determined by the analysis of stable cell lines endogenously expressing the oncogene. Conclusion: EGFRvIII plays an unquestionable role in glioblastomas with high expression of this oncogene. Our data suggests that EGFRvIII importance should not be underestimated even in tumors with relatively low expression of this oncogene. PMID:28123609

Effects of thymidine phosphorylase on tumor aggressiveness and 5-fluorouracil sensitivity in cholangiocarcinoma

PubMed Central

Thanasai, Jongkonnee; Limpaiboon, Temduang; Jearanaikoon, Patcharee; Sripa, Banchob; Pairojkul, Chawalit; Tantimavanich, Srisurang; Miwa, Masanao

2010-01-01

AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy. PMID:20355241

A novel cell model to study the function of the adrenoleukodystrophy-related protein

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

Gueugnon, Fabien; Volodina, Natalia; Taouil, Jaoued Et

2006-03-03

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder due to mutations in the ABCD1 (ALD) gene. ALDRP, the closest homolog of ALDP, has been shown to have partial functional redundancy with ALDP and, when overexpressed, can compensate for the loss-of-function of ALDP. In order to characterize the function of ALDRP and to understand the phenomenon of gene redundancy, we have developed a novel system that allows the controlled expression of the ALDRP-EGFP fusion protein (normal or non-functional mutated ALDRP) using the Tet-On system in H4IIEC3 rat hepatoma cells. The generated stable cell lines express negligible levels of endogenous ALDRP and doxycyclinemore » dosage-dependent levels of normal or mutated ALDRP. Importantly, the ALDRP-EGFP protein is targeted correctly to peroxisome and is functional. The obtained cell lines will be an indispensable tool in our further studies aimed at the resolution of the function of ALDRP to characterize its potential substrates in a natural context.« less

A CRISPR-Based Toolbox for Studying T Cell Signal Transduction

PubMed Central

Chi, Shen; Weiss, Arthur; Wang, Haopeng

2016-01-01

CRISPR/Cas9 system is a powerful technology to perform genome editing in a variety of cell types. To facilitate the application of Cas9 in mapping T cell signaling pathways, we generated a toolbox for large-scale genetic screens in human Jurkat T cells. The toolbox has three different Jurkat cell lines expressing distinct Cas9 variants, including wild-type Cas9, dCas9-KRAB, and sunCas9. We demonstrated that the toolbox allows us to rapidly disrupt endogenous gene expression at the DNA level and to efficiently repress or activate gene expression at the transcriptional level. The toolbox, in combination with multiple currently existing genome-wide sgRNA libraries, will be useful to systematically investigate T cell signal transduction using both loss-of-function and gain-of-function genetic screens. PMID:27057542

Multiplex bioimaging of piRNA molecular pathway-regulated theragnostic effects in a single breast cancer cell using a piRNA molecular beacon.

PubMed

Lee, Youn Jung; Moon, Sung Ung; Park, Min Geun; Jung, Woon Yong; Park, Yong Keun; Song, Sung Kyu; Ryu, Je Gyu; Lee, Yong Seung; Heo, Hye Jung; Gu, Ha Na; Cho, Su Jeong; Ali, Bahy A; Al-Khedhairy, Abdulaziz A; Lee, Ilkyun; Kim, Soonhag

2016-09-01

Recently, PIWI-interacting small non-coding RNAs (piRNAs) have emerged as novel cancer biomarkers candidate because of their high expression level in various cancer types and role in the control of tumor suppressor genes. In this study, a novel breast cancer theragnostics probe based on a single system targeting the piRNA-36026 (piR-36026) molecular pathway was developed using a piR-36026 molecular beacon (MB). The piR-36026 MB successfully visualized endogenous piR-36026 biogenesis, which is highly expressed in MCF7 cells (a human breast cancer cell line), and simultaneously inhibited piR-36026-mediated cancer progression in vitro and in vivo. We discovered two tumor suppressor proteins, SERPINA1 and LRAT, that were directly regulated as endogenous piR-36026 target genes in MCF7 cells. Furthermore, multiplex bioimaging of a single MCF7 cell following treatment with piR-36026 MB clearly visualized the direct molecular interaction of piRNA-36026 with SERPINA1 or LRAT and subsequent molecular therapeutic responses including caspase-3 and PI in the nucleus. Copyright © 2016 Elsevier Ltd. All rights reserved.

1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells

PubMed Central

Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N.; Glenn, Sean T.; Liu, Song; Trump, Donald L.; Johnson, Candace S.

2014-01-01

Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. MiRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253 J and 253J-BV cells express endogenous vitamin D receptor (VDR) which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253 J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. PMID:25263658

Establishment and Characterization of a Telomerase-Immortalized Sheep Trophoblast Cell Line.

PubMed

Zhang, Yufei; Shi, Jing; Liu, Shuying

2016-01-01

The primary sheep trophoblast cells (STCs) have a finite lifespan in culture. This feature limits the scope for long-term in vitro studies with STCs. This study was an attempt to establish and characterize a telomerase-immortalized sheep trophoblast cell line. STCs were isolated and purified by using Percoll and specific immunoaffinity purification, respectively. The purified STCs were transfected with a plasmid carrying sequences of human telomerase reverse transcriptase (hTERT) to create immortalized sheep trophoblast cell line (hTERT-STCs). hTERT-STCs showed a stable expression of hTERT gene, serially passaged for a year, and showed active proliferation without signs of senescence. Cytokeratin 7 (CK-7), secreted human chorionic gonadotrophin subunit β (CG-β), placental lactogen (PL), and endogenous jaagsiekte sheep retrovirus (enJSRV) envelope genes were expressed in hTERT-STCs. Transwell cell invasion assay indicated that hTERT-STCs still possessed the same invasive characteristics as normal primary sheep trophoblast cells. hTERT-STCs could not grow in soft agar and did not develop into tumors in nude mice. In this study, we established a strain of immortalized sheep trophoblast cell line which could be gainfully employed in the future as an experimental model to study trophoblast cells with secretory function, invasive features, and probable biological function of enJSRV envelope genes.

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

PubMed Central

Soares, V da C; Gubits, R M; Feigelson, P; Costantini, F

1987-01-01

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene. Images PMID:2446121

Crossing the LINE toward genomic instability: LINE-1 retrotransposition in cancer

NASA Astrophysics Data System (ADS)

Kemp, Jacqueline; Longworth, Michelle

2015-12-01

Retrotransposons are repetitive DNA sequences that are positioned throughout the human genome. Retrotransposons are capable of copying themselves and mobilizing new copies to novel genomic locations in a process called retrotransposition. While most retrotransposon sequences in the human genome are incomplete and incapable of mobilization, the LINE-1 retrotransposon, which comprises approximately 17% of the human genome, remains active. The disruption of cellular mechanisms that suppress retrotransposon activity is linked to the generation of aneuploidy, a potential driver of tumor development. When retrotransposons insert into a novel genomic region, they have the potential to disrupt the coding sequence of endogenous genes and alter gene expression, which can lead to deleterious consequences for the organism. Additionally, increased LINE-1 copy numbers provide more chances for recombination events to occur between retrotransposons, which can lead to chromosomal breaks and rearrangements. LINE-1 activity is increased in various cancer cell lines and in patient tissues resected from primary tumors. LINE-1 activity also correlates with increased cancer metastasis. This review aims to give a brief overview of the connections between LINE-1 retrotransposition and the loss of genome stability. We will also discuss the mechanisms that repress retrotransposition in human cells and their links to cancer.

The chemical chaperones tauroursodeoxycholic and 4-phenylbutyric acid accelerate thyroid hormone activation and energy expenditure

PubMed Central

da-Silva, Wagner S.; Ribich, Scott; e Drigo, Rafael Arrojo; Castillo, Melany; Patty, Mary-Elizabeth; Bianco, Antonio C.

2011-01-01

Exposure of cell lines endogenously expressing the thyroid hormone activating enzyme type 2 deiodinase (D2) to the chemical chaperones tauroursodeoxycholic acid (TUDCA) or 4-phenylbutiric acid (4-PBA) increases D2 expression, activity and T3 production. In brown adipocytes, TUDCA or 4-PBA induced T3-dependent genes and oxygen consumption (~2-fold), an effect partially lost in D2 knockout cells. In wild type, but not in D2 knockout mice, administration of TUDCA lowered the respiratory quotient, doubled brown adipose tissue D2 activity and normalized the glucose intolerance associated with high fat feeding. Thus, D2 plays a critical role in the metabolic effects of chemical chaperones. PMID:21237159

Rearrangement of Upstream Sequences of the hTERT Gene During Cellular Immortalization

PubMed Central

Zhao, Yuanjun; Wang, Shuwen; Popova, Evgenya Y.; Grigoryev, Sergei A.; Zhu, Jiyue

2010-01-01

Telomerase expression, resulting from transcriptional activation of the hTERT gene, allows cells to acquire indefinite proliferative potential during cellular immortalization and tumorigenesis. However, mechanisms of hTERT gene activation in many immortal cell lines and cancer cells are poorly understood. Here, we report our studies on hTERT activation using genetically related pairs of telomerase-negative (Tel−) and -positive (Tel+) fibroblast lines. First, whereas transiently transfected plasmid reporters did not recapitulate the endogenous hTERT promoter, the promoter in chromosomally integrated bacterial artificial chromosome (BAC) reporters was activated in a subset of Tel+ cells, indicating that activation of the hTERT promoter required native chromatin context and/or distal regulatory elements. Second, the hTERT gene, located near the telomere of chromosome 5p, was translocated in all three Tel+ cell lines but not in their parental pre-crisis cells and Tel− immortal siblings. The breakage points were mapped to regions upstream of the hTERT promoter, indicating that the hTERT gene was the target of these chromosomal rearrangements. In two Tel+ cell lines, translocation of the endogenous hTERT gene appeared to be the major mechanism of its activation as the activity of hTERT promoter in many chromosomally integrated BAC reporters, with intact upstream and downstream neighboring loci, remained relatively low. Therefore, our results suggest that rearrangement of upstream sequences is an important new mechanism of hTERT promoter activation during cellular immortalization. The chromosomal rearrangements likely occurred during cellular crisis and facilitated by telomere dysfunction. Such translocations allowed the hTERT promoter to escape from the native condensed chromatin environment. PMID:19672873

Metabolic characterization of isocitrate dehydrogenase (IDH) mutant and IDH wildtype gliomaspheres uncovers cell type-specific vulnerabilities.

PubMed

Garrett, Matthew; Sperry, Jantzen; Braas, Daniel; Yan, Weihong; Le, Thuc M; Mottahedeh, Jack; Ludwig, Kirsten; Eskin, Ascia; Qin, Yue; Levy, Rachelle; Breunig, Joshua J; Pajonk, Frank; Graeber, Thomas G; Radu, Caius G; Christofk, Heather; Prins, Robert M; Lai, Albert; Liau, Linda M; Coppola, Giovanni; Kornblum, Harley I

2018-01-01

There is considerable interest in defining the metabolic abnormalities of IDH mutant tumors to exploit for therapy. While most studies have attempted to discern function by using cell lines transduced with exogenous IDH mutant enzyme, in this study, we perform unbiased metabolomics to discover metabolic differences between a cohort of patient-derived IDH1 mutant and IDH wildtype gliomaspheres. Using both our own microarray and the TCGA datasets, we performed KEGG analysis to define pathways differentially enriched in IDH1 mutant and IDH wildtype cells and tumors. Liquid chromatography coupled to mass spectrometry analysis with labeled glucose and deoxycytidine tracers was used to determine differences in overall cellular metabolism and nucleotide synthesis. Radiation-induced DNA damage and repair capacity was assessed using a comet assay. Differences between endogenous IDH1 mutant metabolism and that of IDH wildtype cells transduced with the IDH1 (R132H) mutation were also investigated. Our KEGG analysis revealed that IDH wildtype cells were enriched for pathways involved in de novo nucleotide synthesis, while IDH1 mutant cells were enriched for pathways involved in DNA repair. LC-MS analysis with fully labeled 13 C-glucose revealed distinct labeling patterns between IDH1 mutant and wildtype cells. Additional LC-MS tracing experiments confirmed increased de novo nucleotide synthesis in IDH wildtype cells relative to IDH1 mutant cells. Endogenous IDH1 mutant cultures incurred less DNA damage than IDH wildtype cultures and sustained better overall growth following X-ray radiation. Overexpression of mutant IDH1 in a wildtype line did not reproduce the range of metabolic differences observed in lines expressing endogenous mutations, but resulted in depletion of glutamine and TCA cycle intermediates, an increase in DNA damage following radiation, and a rise in intracellular ROS. These results demonstrate that IDH1 mutant and IDH wildtype cells are easily distinguishable metabolically by analyzing expression profiles and glucose consumption. Our results also highlight important differences in nucleotide synthesis utilization and DNA repair capacity that could be exploited for therapy. Altogether, this study demonstrates that IDH1 mutant gliomas are a distinct subclass of glioma with a less malignant, but also therapy-resistant, metabolic profile that will likely require distinct modes of therapy.

Exogenous IFN-beta regulates the RANKL-c-Fos-IFN-beta signaling pathway in the collagen antibody-induced arthritis model.

PubMed

Zhao, Rong; Chen, Ni-Nan; Zhou, Xiao-Wei; Miao, Ping; Hu, Chao-Ying; Qian, Liu; Yu, Qi-Wen; Zhang, Ji-Ying; Nie, Hong; Chen, Xue-hua; Li, Pu; Xu, Rong; Xiao, Lian-Bo; Zhang, Xin; Liu, Jian-Ren; Zhang, Dong-Qing

2014-12-10

Although a variety of drugs have been used to treat the symptoms of rheumatoid arthritis (RA), none of them are able to cure the disease. Interferon β (IFN-β) has pleiotropic effects on RA, but whether it can be used to treat RA remains globally controversial. Thus, in this study we tested the effects of IFN-β on RA patients and on collagen antibody-induced arthritis (CAIA) model mice. The cytokine and auto-antibody expression profiles in the serum and synovial fluid (SF) from RA patients were assessed using enzyme-linked immunosorbent assay (ELISA) and compared with the results from osteoarthritis (OA) patients. Exogenous IFN-β was administered to RA patients and CAIA model mice, and the therapeutic effects were evaluated. Endogenous IFN-β expression in the joint bones of CAIA model mice was evaluated by quantitative real-time PCR (qRT-PCR). The effects of exogenous IFN-β on CAIA model mice were assessed using a clinical scoring system, hematoxylin eosin and safranin-O with fast green counterstain histology, molybdenum target X-ray, and tartrate-resistant acid phosphatase (TRAP) staining. The RANKL-RANK signaling pathway was analyzed using qRT-PCR. The RAW 264.7 cell line was differentiated into osteoclasts with RANKL stimulation and then treated with exogenous IFN-β. The expression of inflammatory cytokines (IFN-γ, IL-17, MMP-3, and RANKL) and auto-antibodies (CII antibodies, RF-IgM, and anti-CCP/GPI) were significantly higher in RA compared with OA patients. After IFN-β intervention, some clinical symptoms in RA patients were partially alleviated, and the expression of IFN-γ, IL-17, MMP-3, and OPG) returned to normal levels. In the CAIA model, the expression of endogenous IFN-β in the joint bones was decreased. After IFN-β administration, the arthritis scores were decreased; synovial inflammation, cartilage, and bone destruction were clearly attenuated; and the expression of c-Fos and NFATc1 were reduced, while RANKL and TRAF6 expression was unchanged. In addition, exogenous IFN-β directly inhibited RANKL-induced osteoclastogenesis. Exogenous IFN-β administration immunomodulates CAIA, may reduce joint inflammation and, perhaps more importantly, bone destruction by inhibiting the RANKL-c-Fos signaling pathway. Exogenous IFN-β intervention should be selectively used on RA patients because it may only be useful for RA patients with low endogenous IFN-β expression.

Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1*

PubMed Central

van de Kooij, Bert; Verbrugge, Inge; de Vries, Evert; Gijsen, Merel; Montserrat, Veronica; Maas, Chiel; Neefjes, Jacques; Borst, Jannie

2013-01-01

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1. PMID:23300075

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

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

Herr, Michael J.; Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38163; Department of Molecular Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163

2014-05-16

Highlights: • CD9 is differentially expressed in human Burkitt’s lymphoma cells. • We found that CD9 expression promotes these cells proliferation. • CD9 expression also increases HDAC activity. • HDAC inhibition decreased both cell proliferation and importantly CD9 expression. • CD9 may dictate HDAC efficacy and play a role in HDAC regulation. - Abstract: Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in twomore » human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.« less

MTA1 regulation of ERβ pathway in salivary gland carcinoma cells

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

Ohshiro, Kazufumi, E-mail: bcmkxo@gwu.edu; Kumar, Rakesh

Abstracts: Although Metastatic-tumor antigen 1 (MTA1) is differentially expressed in metastatic cancer and coregulates the status and activity of nuclear receptors, its role upon estrogen receptor β (ERβ) – a potent tumor suppressor, remains poorly understood. Here we investigated whether MTA1 regulates the expression and functions of ERβ, an ER isoform predominantly expressed in salivary gland cancer cells. We found that the depletion of the endogenous MTA1 in the HSG and HSY salivary duct carcinoma cell lines enhances the expression of ERβ while MTA1 overexpression augmented the expression of ERβ in salivary duct carcinoma cells. Furthermore, MTA1 knockdown inhibited themore » proliferations and invasion of HSG and HSY cells. The noted ERβ downregulation by MTA1 overexpression involves the process of proteasomal degradation, as a proteasome inhibitor could block it. In addition, both MTA1 knockdown and ERβ overexpression attenuated the cell migration and inhibited the ERK1/2 signaling in the both cell lines. These findings imply that MTA1 dysregulation in a subset of salivary gland cancer might promote aggressive phenotypes by compromising the tumor suppressor activity of ERβ, and hence, MTA1-ERβ axis might serve a new therapeutic target for the salivary gland cancer. - Highlights: • MTA1 silencing upregulates ERβ expression in salivary gland carcinoma cells. • MTA1 overexpression downregulates ERβ expression via proteasomal degradation. • Upregulation of ERβ expression inhibits cell migration and ERK signaling. • MTA1 knockdown inhibits cell proliferation and invasion.« less

Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer.

PubMed

German, Sergio D; Campbell, Keith H S; Thornton, Elisabeth; McLachlan, Gerry; Sweetman, Dylan; Alberio, Ramiro

2015-02-01

Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.

Notch3 Signaling-Mediated Melanoma-Endothelial Crosstalk Regulates Melanoma Stem-Like Cell Homeostasis and Niche Morphogenesis

PubMed Central

Hsu, Mei-Yu; Yang, Moon Hee; Schnegg, Caroline I.; Hwang, Soonyean; Ryu, Byungwoo; Alani, Rhoda M.

2016-01-01

Melanoma is among the most virulent cancers, owing to its propensity to metastasize and its resistance to current therapies. The treatment failure is largely attributed to tumor heterogeneity, particularly subpopulations possessing stem cell-like properties, i.e., melanoma stem-like cells (MSLCs). Evidence indicates that the MSLC phenotype is malleable and may be acquired by non-MSLCs through phenotypic switching upon appropriate stimuli, the so–called “dynamic stemness”. Since the phenotypic characteristics and functional integrity of MSLCs depend on their vascular niche, using a two dimensional (2D) melanoma-endothelium co-culture model, where the MSLC niche is recapitulated in vitro, we identified Notch3 signaling pathway as a micro-environmental cue governing MSLC phenotypic plasticity via pathway-specific gene expression arrays. Accordingly, lentiviral shRNA-mediated Notch3 knockdown (KD) in melanoma cell lines exhibiting high levels of endogenous Notch3 led to retarded/abolished tumorigenicity in vivo through both depleting MSLC fractions, evinced by MSLC marker down-regulation (e.g., CD133 and CD271); and impeding the MSLC niche, corroborated by the attenuated tumor angiogenesis as well as vasculogenic mimicry. In contrast, Notch3 KD affected neither tumor growth nor MSLC subsets in a melanoma cell line with relatively low endogenous Notch3 expression. Thus, Notch3 signaling may facilitate MSLC plasticity and niche morphogenesis in a cell context-dependent fashion. Our findings illustrate Notch3 as a molecular switch driving melanoma heterogeneity, and provide the biological rationale for Notch inhibition as a promising therapeutic option. PMID:28165469

Overexpression of SPAG9 in human gastric cancer is correlated with poor prognosis.

PubMed

Miao, Zhi-Feng; Wang, Zhen-Ning; Zhao, Ting-Ting; Xu, Ying-Ying; Wu, Jian-Hua; Liu, Xing-Yu; Xu, Hao; You, Yi; Xu, Hui-Mian

2015-11-01

Sperm associated antigen 9 (SPAG9) protein has been found to play an important role in cancer progression but the involved mechanisms are still obscure. Its clinical significance in human gastric cancers remains unexplored. In the present study, SPAG9 expression was analyzed in 147 gastric cancer specimens. We observed weak staining in normal gastric mucosa and positive staining in 65 out of 147 (44.2 %) cancer samples. Overexpression of SPAG9 correlated with local invasion (p = 0.0101), lymph node metastasis (p = 0.0488), TNM stage (p = 0.0002), and relapse (p = 0.0018). Importantly, SPAG9 overexpression correlated with poor overall survival (p = 0.0008). Furthermore, we performed siRNA knockdown of SPAG9 in HGC-27 cells with high endogenous expression and transfected SPAG9 plasmid in SGC-7901 cell line with low endogenous level. SPAG9 overexpression promoted while its depletion inhibited cell proliferation, cell cycle transition, and invasive cell growth. SPAG9 overxpression also increased chemoresistance to 5--fluorouracil (5-FU) in SGC-7901 cells. Further analysis showed that SPAG9 knockdown downregulated and its overexpression upregulated cyclin D1, MMP9, and p-p38 expression. In conclusion, SPAG9 overexpression in gastric cancer correlates with poor prognosis and contributes to gastric cancer cell proliferation, invasion, and chemoresistance. SPAG9 promotes gastric cancer invasion, possibly through p38-MMP9 signaling pathways.

Effect of cyclophilin A on gene expression in human pancreatic cancer cells.

PubMed

Li, Min; Wang, Hao; Li, Fei; Fisher, William E; Chen, Changyi; Yao, Qizhi

2005-11-01

We previously found that cyclophilin A (CypA) is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. In this study, we further investigated the effect of CypA on gene expression of several key molecules that are involved in pancreatic cancer cell proliferation. Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3) and human pancreatic ductal epithelial (HPDE) cells were used. The messenger RNA (mRNA) levels of CypA, CypB, CD147, neuropilins (NRPs), vascular endothelial growth factor (VEGF), and VEGF receptors upon the treatment of exogenous recombinant human CypA were determined by real-time reverse-transcription polymerase chain reaction. Exogenous human recombinant CypA reduced the mRNA levels of NRP-1 and VEGF, but not endogenous CypA, CypB, and CD147, in Panc-1, MIA PaCa-2, and BxPC-3 cells. In contrast, HPDE cells showed a decrease of endogenous CypA and CD147 mRNA, but not detectable changes of CypB, NRPs, and VEGF mRNA levels upon exogenous CypA treatment. These data show that exogenous CypA downregulates NRP-1 and VEGF expression in pancreatic cancer cells. This effect is different in normal HPDE cells. Thus, soluble CypA may affect cell growth of pancreatic cancer.

Meeting Report: The Role of the Mobilome in Cancer.

PubMed

Ardeljan, Daniel; Taylor, Martin S; Burns, Kathleen H; Boeke, Jef D; Espey, Michael Graham; Woodhouse, Elisa C; Howcroft, Thomas Kevin

2016-08-01

Approximately half of the human genome consists of repetitive sequence attributed to the activities of mobile DNAs, including DNA transposons, RNA transposons, and endogenous retroviruses. Of these, only long interspersed elements (LINE-1 or L1) and sequences copied by LINE-1 remain mobile in our species today. Although cells restrict L1 activity by both transcriptional and posttranscriptional mechanisms, L1 derepression occurs in developmental and pathologic contexts, including many types of cancers. However, we have limited knowledge of the extent and consequences of L1 expression in premalignancies and cancer. Participants in this NIH strategic workshop considered key questions to enhance our understanding of mechanisms and roles the mobilome may play in cancer biology. Cancer Res; 76(15); 4316-9. ©2016 AACR. ©2016 American Association for Cancer Research.

Expression and regulation of human endogenous retrovirus W elements.

PubMed

Li, Fang; Karlsson, Håkan

2016-01-01

Human endogenous retroviruses (HERV) comprise 8% of the human genome and can be classified into at least 31 families. A typical HERV provirus consists of internal gag, pol and env genes, flanked by two long terminal repeats (LTRs). No single provirus is capable of engendering infectious particles. HERV are by nature repetitive and have with few notable exceptions lost their protein-coding capacity. Therefore, HERV have consistently been excluded from array-based expression studies and hence little is known of their expression, regulation, and potential functional significance. An increasing number of studies have, however, observed expression of the W family of HERV in various human tissues and cells, predominantly in placenta. HERV-W LTRs act as promoters in directing transcription of HERV-W members, contribute to their tissue-specific and highly diversified expression pattern. Furthermore, leaky transcription originating from adjacent genes plays a role in the transcription initiation of HERV-W psudoelements. It has been reported that HERV-W elements, including ERVWE1 (the so far only known HERV-W locus harboring a gene (env) functionally adopted by the human host to critically participate in placenta biogenesis), can become transactivated in a range of human non-placental cell-lines during exogenous virus infections. Aberrant expression of HERV-W has been associated with human diseases, such as cancer, multiple sclerosis, and schizophrenia. Based on published reports, transcriptional activities of HERV-W appear to be influenced by several mechanisms; binding of transcription factors to LTR promoters and enhancers outside of LTRs, genetic variation and alteration in DNA methylation and histone modification. Emerging mechanistic studies support the notion that HERV-W represents a potential marker or mediator of environmental exposures (e.g., virus infection) in the development of chronic complex diseases. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Abscisic Acid-Dependent and -Independent Expression of the Carrot Late-Embryogenesis-Abundant-Class Gene Dc3 in Transgenic Tobacco Seedlings1

PubMed Central

Siddiqui, Najeeb U.; Chung, Hwa-Jee; Thomas, Terry L.; Drew, Malcolm C.

1998-01-01

We studied the expression of three promoter 5′ deletion constructs (−218, −599, and −1312) of the LEA (late embryogenesis abundant)-class gene Dc3 fused to β-glucuronidase (GUS), where each construct value refers to the number of base pairs upstream of the transcription start site at which the deletion occurred. The Dc3 gene is noted for its induction by abscisic acid (ABA), but its response to other plant hormones and various environmental stresses has not been reported previously for vegetative cells. Fourteen-day-old transgenic tobacco (Nicotiana tabacum L.) seedlings were exposed to dehydration, hypoxia, salinity, exogenous ethylene, or exogenous methyl jasmonate (MeJa). GUS activity was quantified fluorimetrically and expression was observed by histochemical staining of the seedlings. An increase in GUS activity was observed in plants with constructs −599 and −1312 in response to dehydration and salinity within 6 h of stress, and at 12 h in response to hypoxia. No increase in endogenous ABA was found in any of the three lines, even after 72 h of hypoxia. An ABA-independent increase in GUS activity was observed when endogenous ABA biosynthesis was blocked by fluridone and plants were exposed to 5 μL L−1 ethylene in air or 100 μm MeJa. Virtually no expression was observed in construct −218 in response to dehydration, salinity, or MeJa, but there was a moderate response to ethylene and hypoxia. This suggests that the region between −218 and −599 is necessary for ABA (dehydration and salinity)- and MeJa-dependent expression, whereas ethylene-mediated expression does not require this region of the promoter. PMID:9847092

High level expression of chorismate pyruvate-lyase (UbiC) and HMG-CoA reductase in hairy root cultures of Lithospermum erythrorhizon.

PubMed

Köhle, Annegret; Sommer, Susanne; Yazaki, Kazufumi; Ferrer, Albert; Boronat, Albert; Li, Shu-Ming; Heide, Lutz

2002-08-01

Shikonin, a red naphthoquinone pigment, is produced by cell cultures of Lithospermum erythrorhizon (Boraginaceae). It is biosynthetically derived from two key precursors, 4-hydroxybenzoate (4HB) and geranyldiphosphate (GPP). The bacterial ubiC gene, encoding chorismate pyruvate-lyase (CPL) which converts chorismate to 4-hydroxybenzoate, was expressed in L. erythrorhizon under the control of the strong (ocs)(3)mas-promoter. This introduced an efficient biosynthetic pathway to 4HB, i.e. a one-step reaction from chorismate, in addition to the endogeneous multi-step phenylpropanoid pathway. Feeding experiments with [1,7-(13)C(2)]shikimic acid showed that in the most active transgenic line, 73% of 4HB was synthesized via the genetically introduced pathway. However, there was no correlation between CPL activity and 4HB glucoside or shikonin accumulation in the transgenic lines. HMG-CoA reductase (HMGR) is involved in the biosynthesis of GPP in L. erythrorhizon. Two forms of HMGR1 of Arabidopsis thaliana were expressed in Lithospermum under control of the (ocs)(3)mas promoter. Only moderate increases in enzyme activity were obtained with the complete enzyme, but high activity was achieved using the soluble cytosolic domain of HMGR1. Shikonin accumulation remained unchanged even upon high expression of soluble HMGR.

Downregulation of peptide transporter genes in cell lines transformed with the highly oncogenic adenovirus 12

PubMed Central

1994-01-01

The expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the high oncogenicity of this virus. In primary embryonal fibroblasts from transgenic mice that express both endogenous H-2 genes and a miniature swine class I gene (PD1), Ad12- mediated transformation results in suppression of cell surface expression of all class I antigens. Although class I mRNA levels of PD1 and H-2Db are similar to those in nonvirally transformed cells, recognition of newly synthesized class I molecules by a panel of monoclonal antibodies is impaired, presumably as a result of inefficient assembly and transport of the class I molecules. Class I expression can be partially induced by culturing cells at 26 degrees C, or by coculture of cells with class I binding peptides at 37 degrees C. Analysis of steady state mRNA levels of the TAP1 and TAP2 transporter genes for Ad12-transformed cell lines revealed that they both are significantly reduced, TAP2 by about 100-fold and TAP1 by 5-10-fold. Reconstitution of PD1 and H-2Db, but not H-2Kb, expression is achieved in an Ad12-transformed cell line by stable transfection with a TAP2, but not a TAP1, expression construct. From these data it may be concluded that suppressed expression of peptide transporter genes, especially TAP2, in Ad12-transformed cells inhibits cell surface expression of class I molecules. The failure to fully reconstitute H- 2Db and H-2Kb expression indicates that additional factors are involved in controlling class I gene expression in Ad12-transformed cells. Nevertheless, these results suggest that suppression of peptide transporter genes might be an important mechanism whereby virus- transformed cells escape immune recognition in vivo. PMID:7519239

Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

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

Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.

1991-05-01

Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymicmore » epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells.« less

Expression of CAR in SW480 and HepG2 cells during G1 is associated with cell proliferation

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

Osabe, Makoto; Sugatani, Junko; Global COE Program, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka

Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2more » cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation.« less

Wt-p53 action in human leukaemia cell lines corresponding to different stages of differentiation.

PubMed

Rizzo, M G; Zepparoni, A; Cristofanelli, B; Scardigli, R; Crescenzi, M; Blandino, G; Giuliacci, S; Ferrari, S; Soddu, S; Sacchi, A

1998-05-01

Recent studies support the potential application of the wt-p53 gene in cancer therapy. Expression of exogenous wt-p53 suppresses a variety of leukaemia phenotypes by acting on cell survival, proliferation and/or differentiation. As for tumour gene therapy, the final fate of the neoplastic cells is one of the most relevant points. We examined the effects of exogenous wt-p53 gene expression in several leukaemia cell lines to identify p53-responsive leukaemia. The temperature-sensitive p53Val135 mutant or the human wt-p53 cDNA was transduced in leukaemia cell lines representative of different acute leukaemia FAB subtypes, including M1 (KG1), M2 (HL-60), M3 (NB4), M5 (U937) and M6 (HEL 92.1.7), as well as blast crisis of chronic myelogenous leukaemia (BC-CML: K562, BV173) showing diverse differentiation features. By morphological, molecular and biochemical analyses, we have shown that exogenous wt-p53 gene expression induces apoptosis only in cells corresponding to M1, M2 and M3 of the FAB classification and in BC-CML showing morphological and cytochemical features of undifferentiated blast cells. In contrast, it promotes differentiation in the others. Interestingly, cell responsiveness was independent of the vector used and the status of the endogenous p53 gene.

Enterokinase Enhances Influenza A Virus Infection by Activating Trypsinogen in Human Cell Lines

PubMed Central

Hayashi, Hideki; Kubo, Yoshinao; Izumida, Mai; Takahashi, Etsuhisa; Kido, Hiroshi; Sato, Ko; Yamaya, Mutsuo; Nishimura, Hidekazu; Nakayama, Kou; Matsuyama, Toshifumi

2018-01-01

Cleavage and activation of hemagglutinin (HA) by trypsin-like proteases in influenza A virus (IAV) are essential prerequisites for its successful infection and spread. In host cells, some transmembrane serine proteases such as TMPRSS2, TMPRSS4 and HAT, along with plasmin in the bloodstream, have been reported to cleave the HA precursor (HA0) molecule into its active forms, HA1 and HA2. Some trypsinogens can also enhance IAV proliferation in some cell types (e.g., rat cardiomyoblasts). However, the precise activation mechanism for this process is unclear, because the expression level of the physiological activator of the trypsinogens, the TMPRSS15 enterokinase, is expected to be very low in such cells, with the exception of duodenal cells. Here, we show that at least two variant enterokinases are expressed in various human cell lines, including A549 lung-derived cells. The exogenous expression of these enterokinases was able to enhance the proliferation of IAV in 293T human kidney cells, but the proliferation was reduced by knocking down the endogenous enterokinase in A549 cells. The enterokinase was able to enhance HA processing in the cells, which activated trypsinogen in vitro and in the IAV-infected cells also. Therefore, we conclude that enterokinase plays a role in IAV infection and proliferation by activating trypsinogen to process viral HA in human cell lines. PMID:29629340

Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies

PubMed Central

Sim, Adrian Chong Nyi; Too, Chien Tei; Oo, Min Zin; Lai, Junyun; Eio, Michelle Yating; Song, Zhenying; Srinivasan, Nalini; Tan, Diane Ai Lin; Pang, Shyue Wei; Gan, Shu Uin; Lee, Kok Onn; Loh, Thomas Kwok Seng; Chen, Jianzhu; Chan, Soh Ha; MacAry, Paul Anthony

2013-01-01

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease. PMID:24240815

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

PubMed

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress.

PubMed

Alet, Analía I; Sanchez, Diego H; Cuevas, Juan C; Del Valle, Secundino; Altabella, Teresa; Tiburcio, Antonio F; Marco, Francisco; Ferrando, Alejandro; Espasandín, Fabiana D; González, María E; Ruiz, Oscar A; Carrasco, Pedro

2011-02-01

Polyamines have been globally associated to plant responses to abiotic stress. Particularly, putrescine has been related to a better response to cold and dehydration stresses. It is known that this polyamine is involved in cold tolerance, since Arabidopsis thaliana plants mutated in the key enzyme responsible for putrescine synthesis (arginine decarboxilase, ADC; EC 4.1.1.19) are more sensitive than the wild type to this stress. Although it is speculated that the over-expression of ADC genes may confer tolerance, this is hampered by pleiotropic effects arising from the constitutive expression of enzymes from the polyamine metabolism. Here, we present our work using A. thaliana transgenic plants harboring the ADC gene from oat under the control of a stress-inducible promoter (pRD29A) instead of a constitutive promoter. The transgenic lines presented in this work were more resistant to both cold and dehydration stresses, associated with a concomitant increment in endogenous putrescine levels under stress. Furthermore, the increment in putrescine upon cold treatment correlated with the induction of known stress-responsive genes, and suggested that putrescine may be directly or indirectly involved in ABA metabolism and gene expression.

The human estrogen receptor can regulate exogenous but not endogenous vitellogenin gene promoters in a Xenopus cell line

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

Seiler-Tuyns, A.; Merillat, A.M.; Haefliger, D.N.

Transfection of a human estrogen receptor cDNA expression vector (HEO) into cultured Xenopus kidney cells confers estrogen responsiveness to the recipient cells as demonstrated by the hormone dependent expression of co-transfected Xenopus vitellogenin-CAT chimeric genes. The estrogen stimulation of these vit-CAT genes is dependent upon the presence of the vitellogenin estrogen responsive element (ERE) in their 5{prime} flanking region. Thus, functional human estrogen receptor (hER) can be synthesized in heterologous lower vertebrate cells and can act as a trans-acting regulatory factor that is necessary, together with estradiol, for the induction of the vit-CAT constructs in these cells. In addition, vitellogeninmore » minigenes co-transfected with the HEO expression vector also respond to hormonal stimulation. Their induction is not higher than that of the vit-CAT chimeric genes. It suggests that in the Xenopus kidney cell line B 3.2, the structural parts of the vitellogenin minigenes do not play a role in the induction process. Furthermore, no stabilizing effect of estrogen on vitellogenin mRNA is observed in these cells.« less

Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a-eGFP zebrafish line.

PubMed

Hinfray, Nathalie; Sohm, Frédéric; Caulier, Morgane; Chadili, Edith; Piccini, Benjamin; Torchy, Camille; Porcher, Jean-Marc; Guiguen, Yann; Brion, François

2018-05-15

In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35dpf to reach a high GFP intensity in well-differentiated ovaries at 40dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs. Copyright © 2017 Elsevier Inc. All rights reserved.

Interleukin-8 Promotes Canine Hemangiosarcoma Growth by Regulating the Tumor Microenvironment

PubMed Central

Kim, Jong-Hyuk; Frantz, Aric M.; Anderson, Katie L.; Graef, Ashley J.; Scott, Milcah C.; Robinson, Sally; Sharkey, Leslie C.; O’Brien, Timothy D.; Dickerson, Erin B.; Modiano, Jaime F.

2014-01-01

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggesting that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into “IL-8 high” and “IL-8 low” groups. Genome-wide gene expression profiling showed that samples in the “IL-8 high” tumor group were enriched for genes associated with a “reactive microenvironment,” including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA. PMID:24582862

Interleukin-8 promotes canine hemangiosarcoma growth by regulating the tumor microenvironment.

PubMed

Kim, Jong-Hyuk; Frantz, Aric M; Anderson, Katie L; Graef, Ashley J; Scott, Milcah C; Robinson, Sally; Sharkey, Leslie C; O'Brien, Timothy D; Dickerson, Erin B; Modiano, Jaime F

2014-04-15

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggesting that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into "IL-8 high" and "IL-8 low" groups. Genome-wide gene expression profiling showed that samples in the "IL-8 high" tumor group were enriched for genes associated with a "reactive microenvironment," including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification of small-molecule inhibitors of the colorectal cancer oncogene Krüppel-like factor 5 expression by ultrahigh-throughput screening.

PubMed

Bialkowska, Agnieszka B; Crisp, Melissa; Bannister, Thomas; He, Yuanjun; Chowdhury, Sarwat; Schürer, Stephan; Chase, Peter; Spicer, Timothy; Madoux, Franck; Tian, Chenlu; Hodder, Peter; Zaharevitz, Daniel; Yang, Vincent W

2011-11-01

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium, where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the public domain of NIH, the Molecular Libraries Probe Production Centers Network library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was done in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC(50) < 10 μmol/L for KLF5 inhibition and EC(50) > 10 μmol/L for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized on the basis of computational, Western blot, and cell viability analyses. Both of these compounds, and two newly synthesized structural analogs of CID 5951923, significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results show the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5.

Identification of Small-Molecule Inhibitors of the Colorectal Cancer Oncogene Krüppel-Like Factor 5 Expression by Ultrahigh-Throughput Screening

PubMed Central

Bialkowska, Agnieszka B.; Crisp, Melissa; Bannister, Thomas; He, Yuanjun; Chowdhury, Sarwat; Schürer, Stephan; Chase, Peter; Spicer, Timothy; Madoux, Franck; Tian, Chenlu; Hodder, Peter; Zaharevitz, Daniel; Yang, Vincent W.

2011-01-01

The transcription factor Krüppel-like factor 5 (KLF5) is primarily expressed in the proliferative zone of the mammalian intestinal epithelium where it regulates cell proliferation. Studies showed that inhibition of KLF5 expression reduces proliferation rates in human colorectal cancer cells and intestinal tumor formation in mice. To identify chemical probes that decrease levels of KLF5, we used cell-based ultrahigh-throughput screening (uHTS) to test compounds in the NIH’s public domain, the Molecular Libraries Probe Production Centers Network (MLPCN) library. The primary screen involved luciferase assays in the DLD-1/pGL4.18hKLF5p cell line, which stably expressed a luciferase reporter driven by the human KLF5 promoter. A cytotoxicity counterscreen was performed in the rat intestinal epithelial cell line, IEC-6. We identified 97 KLF5-selective compounds with EC50<10 µM for KLF5 inhibition and EC50>10 µM for IEC-6 cytotoxicity. The two most potent compounds, CIDs (PubChem Compound IDs) 439501 and 5951923, were further characterized based on computational, Western blot, and cell viability analyses. Both of these compounds and two newly-synthesized structural analogs of CID 5951923 significantly reduced endogenous KLF5 protein levels and decreased viability of several colorectal cancer cell lines without any apparent impact on IEC-6 cells. Finally, when tested in the NCI-60 panel of human cancer cell lines, compound CID 5951923 was selectively active against colon cancer cells. Our results demonstrate the feasibility of uHTS in identifying novel compounds that inhibit colorectal cancer cell proliferation by targeting KLF5. PMID:21885866

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

PubMed Central

2010-01-01

Background Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53-activating agents, for the treatment of tumors that retain wild-type p53. PMID:20569441

Antisense reduction of tau in adult mice protects against seizures.

PubMed

DeVos, Sarah L; Goncharoff, Dustin K; Chen, Guo; Kebodeaux, Carey S; Yamada, Kaoru; Stewart, Floy R; Schuler, Dorothy R; Maloney, Susan E; Wozniak, David F; Rigo, Frank; Bennett, C Frank; Cirrito, John R; Holtzman, David M; Miller, Timothy M

2013-07-31

Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS--brain and spinal cord tissue, interstitial fluid, and CSF--while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability.

The Increased Endogenous Sulfur Dioxide Acts as a Compensatory Mechanism for the Downregulated Endogenous Hydrogen Sulfide Pathway in the Endothelial Cell Inflammation

PubMed Central

Zhang, Da; Wang, Xiuli; Tian, Xiaoyu; Zhang, Lulu; Yang, Guosheng; Tao, Yinghong; Liang, Chen; Li, Kun; Yu, Xiaoqi; Tang, Xinjing; Tang, Chaoshu; Zhou, Jing; Kong, Wei; Du, Junbao; Huang, Yaqian; Jin, Hongfang

2018-01-01

Endogenous hydrogen sulfide (H2S) and sulfur dioxide (SO2) are regarded as important regulators to control endothelial cell function and protect endothelial cell against various injuries. In our present study, we aimed to investigate the effect of endogenous H2S on the SO2 generation in the endothelial cells and explore its significance in the endothelial inflammation in vitro and in vivo. The human umbilical vein endothelial cell (HUVEC) line (EA.hy926), primary HUVECs, primary rat pulmonary artery endothelial cells (RPAECs), and purified aspartate aminotransferase (AAT) protein from pig heart were used for in vitro experiments. A rat model of monocrotaline (MCT)-induced pulmonary vascular inflammation was used for in vivo experiments. We found that endogenous H2S deficiency caused by cystathionine-γ-lyase (CSE) knockdown increased endogenous SO2 level in endothelial cells and enhanced the enzymatic activity of AAT, a major SO2 synthesis enzyme, without affecting the expressions of AAT1 and AAT2. While H2S donor could reverse the CSE knockdown-induced increase in the endogenous SO2 level and AAT activity. Moreover, H2S donor directly inhibited the activity of purified AAT protein, which was reversed by a thiol reductant DTT. Mechanistically, H2S donor sulfhydrated the purified AAT1/2 protein and rescued the decrease in the sulfhydration of AAT1/2 protein in the CSE knockdown endothelial cells. Furthermore, an AAT inhibitor l-aspartate-β-hydroxamate (HDX), which blocked the upregulation of endogenous SO2/AAT generation induced by CSE knockdown, aggravated CSE knockdown-activated nuclear factor-κB pathway in the endothelial cells and its downstream inflammatory factors including ICAM-1, TNF-α, and IL-6. In in vivo experiment, H2S donor restored the deficiency of endogenous H2S production induced by MCT, and reversed the upregulation of endogenous SO2/AAT pathway via sulfhydrating AAT1 and AAT2. In accordance with the results of the in vitro experiment, HDX exacerbated the pulmonary vascular inflammation induced by the broken endogenous H2S production in MCT-treated rat. In conclusion, for the first time, the present study showed that H2S inhibited endogenous SO2 generation by inactivating AAT via the sulfhydration of AAT1/2; and the increased endogenous SO2 generation might play a compensatory role when H2S/CSE pathway was downregulated, thereby exerting protective effects in endothelial inflammatory responses in vitro and in vivo. PMID:29760703

lin-4 and the NRDE pathway are required to activate a transgenic lin-4 reporter but not the endogenous lin-4 locus in C. elegans.

PubMed

Jiao, Alan L; Foster, Daniel J; Dixon, Julia; Slack, Frank J

2018-01-01

As the founding member of the microRNA (miRNA) gene family, insights into lin-4 regulation and function have laid a conceptual foundation for countless miRNA-related studies that followed. We previously showed that a transcriptional lin-4 reporter in C. elegans was positively regulated by a lin-4-complementary element (LCE), and by lin-4 itself. In this study, we sought to (1) identify additional factors required for lin-4 reporter expression, and (2) validate the endogenous relevance of a potential positive autoregulatory mechanism of lin-4 expression. We report that all four core nuclear RNAi factors (nrde-1, nrde-2, nrde-3 and nrde-4), positively regulate lin-4 reporter expression. In contrast, endogenous lin-4 levels were largely unaffected in nrde-2;nrde-3 mutants. Further, an endogenous LCE deletion generated by CRISPR-Cas9 revealed that the LCE was also not necessary for the activity of the endogenous lin-4 promoter. Finally, mutations in mature lin-4 did not reduce primary lin-4 transcript levels. Taken together, these data indicate that under growth conditions that reveal effects at the transgenic locus, a direct, positive autoregulatory mechanism of lin-4 expression does not occur in the context of the endogenous lin-4 locus.

Activation of Wnt signaling pathway by human papillomavirus E6 and E7 oncogenes in HPV16-positive oropharyngeal squamous carcinoma cells.

PubMed

Rampias, Theodore; Boutati, Eleni; Pectasides, Eirini; Sasaki, Clarence; Kountourakis, Panteleimon; Weinberger, Paul; Psyrri, Amanda

2010-03-01

We sought to determine the role of human papillomavirus (HPV) E6 and E7 oncogenes in nuclear beta-catenin accumulation, a hallmark of activated canonical Wnt signaling pathway. We used HPV16-positive oropharyngeal cancer cell lines 147T and 090, HPV-negative cell line 040T, and cervical cell lines SiHa (bearing integrated HPV16) and HeLa (bearing integrated HPV18) to measure the cytoplasmic and nuclear beta-catenin levels and the beta-catenin/Tcf transcriptional activity before and after E6/E7 gene silencing. Repression of HPV E6 and E7 genes induced a substantial reduction in nuclear beta-catenin levels. Luciferase assay showed that transcriptional activation of Tcf promoter by beta-catenin was lower after silencing. The protein levels of beta-catenin are tightly regulated by the ubiquitin/proteasome system. We therefore performed expression analysis of regulators of beta-catenin degradation and nuclear transport and showed that seven in absentia homologue (Siah-1) mRNA and protein levels were substantially upregulated after E6/E7 repression. Siah-1 protein promotes the degradation of beta-catenin through the ubiquitin/proteasome system. To determine whether Siah-1 is important for the proteasomal degradation of beta-catenin in HPV16-positive oropharyngeal cancer cells, we introduced a Siah-1 expression vector into 147T and 090 cells and found substantial reduction of endogenous beta-catenin in these cells. Thus, E6 and E7 are involved in beta-catenin nuclear accumulation and activation of Wnt signaling in HPV-induced cancers. In addition, we show the significance of the endogenous Siah-1-dependent ubiquitin/proteasome pathway for beta-catenin degradation and its regulation by E6/E7 viral oncoproteins in HPV16-positive oropharyngeal cancer cells.

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells

PubMed Central

Peters, Haley L.; Tripathi, Satyendra C.; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R.; St. John, Lisa S.; Federico, Lorenzo; Meraz, Ismail M.; Roth, Jack A.; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L.; Heymach, John V.; Swisher, Stephen G.; Bernantchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J.

2017-01-01

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these re-invigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAM were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTL from healthy donors that had been expanded against select peptides. Finally, CTL specific for serine proteases–induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. PMID:28254787

Cholecystokinin 1 Receptor - A Unique G Protein-Coupled Receptor Activated by Singlet Oxygen (GPCR-ABSO).

PubMed

Jiang, Hong Ning; Li, Yuan; Jiang, Wen Yi; Cui, Zong Jie

2018-01-01

Plasma membrane-delimited generation of singlet oxygen by photodynamic action with photosensitizer sulfonated aluminum phthalocyanine (SALPC) activates cholecystokinin 1 receptor (CCK1R) in pancreatic acini. Whether CCK1R retains such photooxidative singlet oxygen activation properties in other environments is not known. Genetically encoded protein photosensitizers KillerRed or mini singlet oxygen generator (miniSOG) were expressed in pancreatic acinar tumor cell line AR4-2J, CCK1R, KillerRed or miniSOG were expressed in HEK293 or CHO-K1 cells. Cold light irradiation (87 mW⋅cm -2 ) was applied to photosensitizer-expressing cells to examine photodynamic activation of CCK1R by Fura-2 fluorescent calcium imaging. When CCK1R was transduced into HEK293 cells which lack endogenous CCK1R, photodynamic action with SALPC was found to activate CCK1R in CCK1R-HEK293 cells. When KillerRed or miniSOG were transduced into AR4-2J which expresses endogenous CCK1R, KillerRed or miniSOG photodynamic action at the plasma membrane also activated CCK1R. When fused KillerRed-CCK1R was transduced into CHO-K1 cells, light irradiation activated the fused CCK1R leading to calcium oscillations. Therefore KillerRed either expressed independently, or fused with CCK1R can both activate CCK1R photodynamically. It is concluded that photodynamic singlet oxygen activation is an intrinsic property of CCK1R, independent of photosensitizer used, or CCK1R-expressing cell types. Photodynamic singlet oxygen CCK1R activation after transduction of genetically encoded photosensitizer in situ may provide a convenient way to verify intrinsic physiological functions of CCK1R in multiple CCK1R-expressing cells and tissues, or to actuate CCK1R function in CCK1R-expressing and non-expressing cell types after transduction with fused KillerRed-CCK1R.

Regulation of Human γδ T Cells by BTN3A1 Protein Stability and ATP-Binding Cassette Transporters

PubMed Central

Rhodes, David A.; Chen, Hung-Chang; Williamson, James C.; Hill, Alfred; Yuan, Jack; Smith, Sam; Rhodes, Harriet; Trowsdale, John; Lehner, Paul J.; Herrmann, Thomas; Eberl, Matthias

2018-01-01

Activation of human Vγ9/Vδ2 T cells by “phosphoantigens” (pAg), the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and the endogenous isoprenoid intermediate isopentenyl pyrophosphate, requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30.2 domain of BTN3A1. To approach these problems, we studied two HLA haplo-identical HeLa cell lines, termed HeLa-L and HeLa-M, which showed marked differences in pAg-dependent stimulation of Vγ9/Vδ2 T cells. Levels of IFN-γ secretion by Vγ9/Vδ2 T cells were profoundly increased by pAg loading, or by binding of the pan-BTN3A specific agonist antibody CD277 20.1, in HeLa-M compared to HeLa-L cells. IL-2 production from a murine hybridoma T cell line expressing human Vγ9/Vδ2 T cell receptor (TCR) transgenes confirmed that the differential responsiveness to HeLa-L and HeLa-M was TCR dependent. By tissue typing, both HeLa lines were shown to be genetically identical and full-length transcripts of the three BTN3A isoforms were detected in equal abundance with no sequence variation. Expression of BTN3A and interacting molecules, such as periplakin or RhoB, did not account for the functional variation between HeLa-L and HeLa-M cells. Instead, the data implicate a checkpoint controlling BTN3A1 stability and protein trafficking, acting at an early time point in its maturation. In addition, plasma membrane profiling was used to identify proteins upregulated in HMB-PP-treated HeLa-M. ABCG2, a member of the ATP-binding cassette (ABC) transporter family was the most significant candidate, which crucially showed reduced expression in HeLa-L. Expression of a subset of ABC transporters, including ABCA1 and ABCG1, correlated with efficiency of T cell activation by cytokine secretion, although direct evidence of a functional role was not obtained by knockdown experiments. Our findings indicate a link between members of the ABC protein superfamily and the BTN3A-dependent activation of γδ T cells by endogenous and exogenous pAg. PMID:29670629

The Interactive Effects of Transgenically Overexpressed 1Ax1 with Various HMW-GS Combinations on Dough Quality by Introgression of Exogenous Subunits into an Elite Chinese Wheat Variety

PubMed Central

Zhang, Jian; Lei, Qian; Meng, Dandan; Ma, Fengyun; Hu, Wei; Chen, Mingjie; Chang, Junli; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2013-01-01

Seed storage proteins in wheat endosperm, particularly high-molecular-weight glutenin subunits (HMW-GS), are primary determinants of dough properties, and affect both end-use quality and grain utilization of wheat (Triticum aestivum L). In order to investigate the interactive effects between the transgenically overexpressed 1Ax1 subunit with different HMW-GS on dough quality traits, we developed a set of 8 introgression lines (ILs) overexpressing the transgenic HMW-glutenin subunit 1Ax1 by introgression of this transgene from transgenic line B102-1-2/1 into an elite Chinese wheat variety Chuanmai107 (C107), using conventional crossing and backcrossing breeding technique. The donor C107 strain lacks 1Ax1 but contains the HMW-GS pairs 1Dx2+1Dy12 and 1Bx7+1By9. The resultant ILs showed robust and stable expression of 1Ax1 even after five generations of self-pollination, and crossing/backcrossing three times. In addition, overexpression of 1Ax1 was compensated by the endogenous gluten proteins. All ILs exhibited superior agronomic performance when compared to the transgenic parent line, B102-1-2/1. Mixograph results demonstrated that overexpressed 1Ax1 significantly improved dough strength, resistance to extension and over-mixing tolerance, in the targeted wheat cultivar C107. Further, comparisons among the ILs showed the interactive effects of endogenous subunits on dough properties when 1Ax1 was overexpressed: subunit pair 17+18 contributed to increased over-mixing tolerance of the dough; expression of the Glu-D1 allele maintained an appropriate balance between x-type and y-type subunits and thereby improved dough quality. It is consistent with ILs C4 (HMW-GS are 1, 17+18, 2+12) had the highest gluten index and Zeleny sedimentation value. This study demonstrates that wheat quality could be improved by using transgenic wheat overexpressing HMW-GS and the feasibility of using such transgenic lines in wheat quality breeding programs. PMID:24167625

Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

PubMed Central

Muto, Machiko; Henry, Ryan E; Mayfield, Stephen P

2009-01-01

Background Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct. Results The luciferase from the fusion protein accumulated to significantly higher levels than luciferase expressed alone. By eliminating the endogenous Rubisco large subunit gene (rbcL), we achieved a further increase in luciferase accumulation with respect to luciferase expression in the WT background. Importantly, near-wild type levels of functional Rubisco holoenzyme were generated following the proteolytic removal of the fused luciferase, while luciferase activity for the fusion protein was almost ~33 times greater than luciferase expressed alone. These data demonstrate the utility of using fusion proteins to enhance recombinant protein accumulation in algal chloroplasts, and also show that engineered proteolytic processing sites can be used to liberate the exogenous protein from the endogenous fusion partner, allowing for the purification of the intended mature protein. Conclusion These results demonstrate the utility of fusion proteins in algal chloroplast as a method to increase accumulation of recombinant proteins that are difficult to express. Since Rubisco is ubiquitous to land plants and green algae, this strategy may also be applied to higher plant transgenic expression systems. PMID:19323825

Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance.

PubMed

Hamdollah Zadeh, Maryam A; Amin, Elianna M; Hoareau-Aveilla, Coralie; Domingo, Enric; Symonds, Kirsty E; Ye, Xi; Heesom, Katherine J; Salmon, Andrew; D'Silva, Olivia; Betteridge, Kai B; Williams, Ann C; Kerr, David J; Salmon, Andrew H J; Oltean, Sebastian; Midgley, Rachel S; Ladomery, Michael R; Harper, Steven J; Varey, Alexander H R; Bates, David O

2015-01-01

The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A165b. Whereas flTIA-1 selectively bound VEGF-A165 mRNA and increased translation of VEGF-A165b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo.

PubMed

Vaiphei, S Thangminlal; Keppen, Joshua; Nongrum, Saibadaiahun; Chaubey, R C; Kma, L; Sharan, R N

2015-01-01

In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving (60)Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of (60)Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min(-1) at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Genetic tracing of the gustatory and trigeminal neural pathways originating from T1R3-expressing taste receptor cells and solitary chemoreceptor cells.

PubMed

Ohmoto, Makoto; Matsumoto, Ichiro; Yasuoka, Akihito; Yoshihara, Yoshihiro; Abe, Keiko

2008-08-01

We established transgenic mouse lines expressing a transneuronal tracer, wheat germ agglutinin (WGA), under the control of mouse T1R3 gene promoter/enhancer. In the taste buds, WGA transgene was faithfully expressed in T1R3-positive sweet/umami taste receptor cells. WGA protein was transferred not laterally to the synapse-bearing, sour-responsive type III cells in the taste buds but directly to a subset of neurons in the geniculate and nodose/petrosal ganglia, and further conveyed to a rostro-central region of the nucleus of solitary tract. In addition, WGA was expressed in solitary chemoreceptor cells in the nasal epithelium and transferred along the trigeminal sensory pathway to the brainstem neurons. The solitary chemoreceptor cells endogenously expressed T1R3 together with bitter taste receptors T2Rs. This result shows an exceptional signature of receptor expression. Thus, the t1r3-WGA transgenic mice revealed the sweet/umami gustatory pathways from taste receptor cells and the trigeminal neural pathway from solitary chemoreceptor cells.

Parvovirus-Derived Endogenous Viral Elements in Two South American Rodent Genomes

PubMed Central

2014-01-01

We describe endogenous viral elements (EVEs) derived from parvoviruses (family Parvoviridae) in the genomes of the long-tailed chinchilla (Chinchilla lanigera) and the degu (Octodon degus). The novel EVEs include dependovirus-related elements and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In the degu, one dependovirus-derived EVE was found to carry an intact reading frame and was differentially expressed in vivo, with increased expression in the liver. PMID:25078696

Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

PubMed

Hrycay, E G; Bandiera, S M

2009-12-01

The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

Malat1 regulates serum response factor through miR-133 as a competing endogenous RNA in myogenesis.

PubMed

Han, Xiaorui; Yang, Feng; Cao, Huiqing; Liang, Zicai

2015-07-01

Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is an example of a functional long noncoding RNA involved in many biologic processes. However, the mechanisms for Malat1 in myogenesis are unclear. Serum response factor (SRF) is a pivotal transcription factor for muscle proliferation and differentiation and is reported to be a target gene for muscle-specific microRNA-133 (miR-133). In this study, we initially found that silencing Malat1 in the mouse myoblast C2C12 cell line inhibited myocyte differentiation and decreased Srf at both the RNA and protein levels. Srf silencing decreased Malat1 expression as well. Further study revealed that Malat1 contained an miR-133 functional target site, and the interplay between Malat1 and Srf was miR-133 dependent. We demonstrated that Malat1 modulates Srf through miR-133 as a competing endogenous RNA and established a novel connection among Malat1, miR-133, and Srf in myoblast differentiation. © FASEB.

Pleiotrophin prevents cocaine-induced toxicity in vitro.

PubMed

Gramage, Esther; Alguacil, Luis F; Herradon, Gonzalo

2008-10-24

Pleiotrophin is a cytokine involved in differentiation, survival and repair processes in the central nervous system. Pleiotrophin is upregulated in the brain after administration of different drugs of abuse, thus suggesting a protective role of this cytokine on drug-induced toxicity. We have tested this hypothesis in vitro using NG108-15 cells, a line widely used for neurotoxicity studies. It was found that pleiotrophin (3 and 6 microM) significantly prevents cocaine (5 mM)-induced cytotoxicity as measured by the neutral red test. Similar results were obtained in PC12 cells, which were found to endogenously express both pleiotrophin and its main target, receptor protein tyrosine phosphatase (RPTP) beta/zeta. Blockade of pleiotrophin signaling using anti-pleiotrophin antibodies (2 microg/ml) did not potentiate cocaine-induced toxicity; interestingly, incubation of PC12 cells only with anti-pleiotrophin antibodies significantly reduced cellular viability, thus suggesting an important role of endogenous pleiotrophin signaling in cell survival. The data suggest that pleiotrophin overexpression in response to drugs of abuse may be relevant to prevent drug-induced toxicity.

HTLV-I Tax transrepresses the human c-Myb promoter independently of its interaction with CBP or p300.

PubMed

Nicot, C; Mahieux, R; Opavsky, R; Cereseto, A; Wolff, L; Brady, J N; Franchini, G

2000-04-20

The c-Myb proto-oncogene is preferentially expressed in hematopoietic lineages, and highly expressed in several leukemia types. The Human T-cell Leukemia Virus Type I (HTLV-I) is the etiological agent of Adult T-cell Leukemia/Lymphoma (ATLL). A previous report suggested that Tax, the viral transactivator, is able to suppress the transactivation potential of c-Myb protein by competing for recruitment of CBP. We tested whether such a competition could affect transcription from the c-Myb promoter in Tax expressing T-cells. Using several c-Myb promoter reporter constructs carrying mutations in various regions, we demonstrate that Tax suppression of c-Myb transactivation results in transrepression of the c-Myb promoter through the Myb responsive elements in Jurkat T-cells. The ability of Tax mutants M22, M47 and V89A to interact with the full-length CBP and p300 proteins in vitro, and their ability to repress the c-Myb promoter, was then evaluated. Although both M47 and M22 bind to CBP and p300 to a similar extent, only M47 was able to repress the c-Myb promoter, suggesting that competition for CBP/p300 binding was not the mechanism underlying Tax's effect. This concept was further supported by the fact that the Tax mutant V89A transrepresses the c-Myb promoter efficiently in spite of an impaired binding to CBP and p300. Therefore, Tax-mediated repression of the c-Myb promoter appears to be independent from a direct competition between c-Myb and Tax for recruitment of CBP/p300. Interestingly, a decreased transcription from the endogenous c-Myb promoter was observed in several HTLV-I transformed T-cell lines. Finally, the ability of Tax to directly repress the endogenous c-Myb promoter was demonstrated in a Jurkat cell line stably transfected with a tax gene driven by a cadmium-inducible promoter.

miRNA-558 promotes gastric cancer progression through attenuating Smad4-mediated repression of heparanase expression.

PubMed

Zheng, Liduan; Jiao, Wanju; Song, Huajie; Qu, Hongxia; Li, Dan; Mei, Hong; Chen, Yajun; Yang, Feng; Li, Huanhuan; Huang, Kai; Tong, Qiangsong

2016-09-29

Previous studies have indicated that as the only mammalian endo-β-D-glucuronidase, heparanase (HPSE) is up-regulated and associated with poor prognosis in gastric cancer, while the underlying mechanisms still remain to be determined. Herein, through integrative analysis of public datasets, we found microRNA-558 (miR-558) and SMAD family member 4 (Smad4) as the crucial transcription regulators of HPSE expression in gastric cancer, with their adjacent target sites within the promoter of HPSE. We identified that endogenous miR-558 activated the transcription and expression of HPSE in gastric cancer cell lines. In contrast, Smad4 suppressed the nascent transcription and expression of HPSE via directly binding to its promoter. Mechanistically, miR-558 recognized its complementary site within HPSE promoter to decrease the binding of Smad4 in an Argonaute 1-dependent manner. Ectopic expression or knockdown experiments indicated that miR-558 promoted the in vitro and in vivo tumorigenesis and aggressiveness of gastric cancer cell lines via attenuating Smad4-mediated repression of HPSE expression. In clinical gastric cancer specimens, up-regulation of miR-558 and down-regulation of Smad4 were positively correlated with HPSE expression. Kaplan-Meier survival analysis revealed that miR-558 and Smad4 were associated with unfavourable and favourable outcome of gastric cancer patients, respectively. Therefore, these findings demonstrate that miR-558 facilitates the progression of gastric cancer through directly targeting the HPSE promoter to attenuate Smad4-mediated repression of HPSE expression.

miRNA-558 promotes gastric cancer progression through attenuating Smad4-mediated repression of heparanase expression

PubMed Central

Zheng, Liduan; Jiao, Wanju; Song, Huajie; Qu, Hongxia; Li, Dan; Mei, Hong; Chen, Yajun; Yang, Feng; Li, Huanhuan; Huang, Kai; Tong, Qiangsong

2016-01-01

Previous studies have indicated that as the only mammalian endo-β-D-glucuronidase, heparanase (HPSE) is up-regulated and associated with poor prognosis in gastric cancer, while the underlying mechanisms still remain to be determined. Herein, through integrative analysis of public datasets, we found microRNA-558 (miR-558) and SMAD family member 4 (Smad4) as the crucial transcription regulators of HPSE expression in gastric cancer, with their adjacent target sites within the promoter of HPSE. We identified that endogenous miR-558 activated the transcription and expression of HPSE in gastric cancer cell lines. In contrast, Smad4 suppressed the nascent transcription and expression of HPSE via directly binding to its promoter. Mechanistically, miR-558 recognized its complementary site within HPSE promoter to decrease the binding of Smad4 in an Argonaute 1-dependent manner. Ectopic expression or knockdown experiments indicated that miR-558 promoted the in vitro and in vivo tumorigenesis and aggressiveness of gastric cancer cell lines via attenuating Smad4-mediated repression of HPSE expression. In clinical gastric cancer specimens, up-regulation of miR-558 and down-regulation of Smad4 were positively correlated with HPSE expression. Kaplan–Meier survival analysis revealed that miR-558 and Smad4 were associated with unfavourable and favourable outcome of gastric cancer patients, respectively. Therefore, these findings demonstrate that miR-558 facilitates the progression of gastric cancer through directly targeting the HPSE promoter to attenuate Smad4-mediated repression of HPSE expression. PMID:27685626

Induction of PD-L1 Expression by the EML4-ALK Oncoprotein and Downstream Signaling Pathways in Non-Small Cell Lung Cancer.

PubMed

Ota, Keiichi; Azuma, Koichi; Kawahara, Akihiko; Hattori, Satoshi; Iwama, Eiji; Tanizaki, Junko; Harada, Taishi; Matsumoto, Koichiro; Takayama, Koichi; Takamori, Shinzo; Kage, Masayoshi; Hoshino, Tomoaki; Nakanishi, Yoichi; Okamoto, Isamu

2015-09-01

Therapies targeted to the immune checkpoint mediated by PD-1 and PD-L1 show antitumor activity in a subset of patients with non-small cell lung cancer (NSCLC). We have now examined PD-L1 expression and its regulation in NSCLC positive for the EML4-ALK fusion gene. The expression of PD-L1 at the protein and mRNA levels in NSCLC cell lines was examined by flow cytometry and by reverse transcription and real-time PCR analysis, respectively. The expression of PD-L1 in 134 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. The PD-L1 expression level was higher in NSCLC cell lines positive for EML4-ALK than in those negative for the fusion gene. Forced expression of EML4-ALK in Ba/F3 cells markedly increased PD-L1 expression, whereas endogenous PD-L1 expression in EML4-ALK-positive NSCLC cells was attenuated by treatment with the specific ALK inhibitor alectinib or by RNAi with ALK siRNAs. Furthermore, expression of PD-L1 was downregulated by inhibitors of the MEK-ERK and PI3K-AKT signaling pathways in NSCLC cells positive for either EML4-ALK or activating mutations of the EGFR. Finally, the expression level of PD-L1 was positively associated with the presence of EML4-ALK in NSCLC specimens. Our findings that both EML4-ALK and mutant EGFR upregulate PD-L1 by activating PI3K-AKT and MEK-ERK signaling pathways in NSCLC reveal a direct link between oncogenic drivers and PD-L1 expression. ©2015 American Association for Cancer Research.

Tissue transglutaminase regulates focal adhesion kinase/AKT activation by modulating PTEN expression in pancreatic cancer cells.

PubMed

Verma, Amit; Guha, Sushovan; Wang, Huamin; Fok, Jansina Y; Koul, Dimpy; Abbruzzese, James; Mehta, Kapil

2008-04-01

Pancreatic ductal adenocarcinoma (PDAC) progresses rapidly and exhibits profound resistance to treatment. We recently reported that a great majority of PDAC tumors and tumor cell lines express elevated levels of tissue transglutaminase (TG2). Here, we provide first evidence that TG2 expression in PDAC cells results in constitutive activation of focal adhesion kinase/AKT by modulating the expression of the tumor suppressor phosphatase PTEN. Using PDAC cell lines, we determined the effect of TG2 overexpression on PTEN stability and functions. We confirmed the correlation between TG2 expression and PTEN levels in a few (n=51) PDAC tumor samples. We observed that expression of TG2 is inversely correlated with PTEN expression in PDAC cells. Ectopic expression of TG2 inhibited PTEN phosphorylation and promoted its degradation by ubiquitin-proteasomal pathway. Conversely, down-regulation of TG2 by small interfering RNA up-regulated PTEN expression. Clinical relevance of these results was evident in an athymic nude mouse model where down-regulation of endogenous TG2 caused a significant retardation in PDAC xenograft growth. Importantly, the analysis of 51 tumor samples from patients with stage II PDAC revealed that overexpression of TG2 was associated with loss of PTEN expression (P=0.023; odds ratio, 4.1). In multivariate analysis, TG2-mediated loss of PTEN was a prognostic factor for overall survival in patients with stage II pancreatic ductal carcinoma independent of tumor stage/lymph node status and tumor differentiation (P=0.01). TG2 expression in PDAC promotes degradation of PTEN by ubiquitin-proteasomal pathway and results in constitutive activation of focal adhesion kinase/AKT cell survival signaling.

Overexpressing Ferredoxins in Chlamydomonas reinhardtii Increase Starch and Oil Yields and Enhance Electric Power Production in a Photo Microbial Fuel Cell

PubMed Central

Huang, Li-Fen; Lin, Ji-Yu; Pan, Kui-You; Huang, Chun-Kai; Chu, Ying-Kai

2015-01-01

Ferredoxins (FDX) are final electron carrier proteins in the plant photosynthetic pathway, and function as major electron donors in diverse redox-driven metabolic pathways. We previously showed that overexpression of a major constitutively expressed ferredoxin gene PETF in Chlamydomonas decreased the reactive oxygen species (ROS) level and enhanced tolerance to heat stress. In addition to PETF, an endogenous anaerobic induced FDX5 was overexpressed in transgenic Chlamydomonas lines here to address the possible functions of FDX5. All the independent FDX transgenic lines showed decreased cellular ROS levels and enhanced tolerance to heat and salt stresses. The transgenic Chlamydomonas lines accumulated more starch than the wild-type line and this effect increased almost three-fold in conditions of nitrogen depletion. Furthermore, the lipid content was higher in the transgenic lines than in the wild-type line, both with and without nitrogen depletion. Two FDX-overexpressing Chlamydomonas lines were assessed in a photo microbial fuel cell (PMFC); power density production by the transgenic lines was higher than that of the wild-type cells. These findings suggest that overexpression of either PETF or FDX5 can confer tolerance against heat and salt stresses, increase starch and oil production, and raise electric power density in a PMFC. PMID:26287179

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

PubMed Central

Roberts, Brock; Haupt, Amanda; Tucker, Andrew; Grancharova, Tanya; Arakaki, Joy; Fuqua, Margaret A.; Nelson, Angelique; Hookway, Caroline; Ludmann, Susan A.; Mueller, Irina A.; Yang, Ruian; Horwitz, Rick; Rafelski, Susanne M.; Gunawardane, Ruwanthi N.

2017-01-01

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1–4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line–generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. PMID:28814507

LINE-1 Retroelements Complexed and Inhibited by Activation Induced Cytidine Deaminase

PubMed Central

Metzner, Mirjam; Jäck, Hans-Martin; Wabl, Matthias

2012-01-01

LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus. PMID:23133680

Recombinant interferon-gamma secreted by Chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium.

PubMed

Mols, J; Peeters-Joris, C; Wattiez, R; Agathos, S N; Schneider, Y-J

2005-01-01

Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-gamma (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and -2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

PubMed Central

Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A.; Esko, Jeffrey D.; Linhardt, Robert J.; Sharfstein, Susan T.

2012-01-01

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS / heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS / heparin biosynthesis might be necessary. PMID:22326251

Long interspersed nuclear element-1 retroelements are expressed in patients with systemic autoimmune disease and induce type I interferon

PubMed Central

Mavragani, Clio P.; Sagalovskiy, Irina; Guo, Qiu; Nezos, Adrianos; Kapsogeorgou, Efstathia K.; Lu, Pin; Zhou, Jun Liang; Kirou, Kyriakos A.; Seshan, Surya V.; Moutsopoulos, Haralampos M.; Crow, Mary K.

2016-01-01

Objective Increased type I interferon (IFN-I) and a broad signature of IFN-I-induced gene transcripts are observed in patients with SLE and other systemic autoimmune diseases. To identify disease-relevant triggers of the IFN-I pathway we investigated whether endogenous virus-like genomic repeat elements, normally silent, might be expressed in patients with systemic autoimmune disease, activate an innate immune response and induce IFN-I. Methods Expression of IFN-I and long interspersed nuclear element-1 (LINE-1; L1) was studied in kidney tissue from lupus patients and minor salivary gland (MSG) tissue from patients with primary Sjogren’s syndrome (SS) by PCR, western blot and immunohistochemistry. Induction of IFN-I by L1 was investigated by transfection of plasmacytoid dendritic cells (pDCs) or monocytes with an L1-encoding plasmid or L1 RNA. Involvement of innate immune pathways and altered L1 methylation were assessed. Results L1 mRNA transcripts were increased in lupus nephritis kidneys and in MSG from SS patients and correlated with IFN-I expression and L1 DNA demethylation. L1 open reading frame 1/p40 protein and IFNβ were expressed in MSG ductal epithelial cells and in lupus kidneys, and IFNα was detected in infiltrating pDCs. Transfection of pDCs or monocytes with L1-encoding DNA or RNA induced IFN-I. Inhibition of TLR7/8 reduced L1 induction of IFNα in pDCs and an inhibitor of IKKε/TBK1 abrogated induction of IFN-I by L1 RNA in monocytes. Conclusion L1 genomic repeat elements represent endogenous nucleic acid triggers of the IFN-I pathway in SLE and SS and may contribute to initiation or amplification of autoimmune disease. PMID:27338297

R1, a novel repressor of the human monoamine oxidase A.

PubMed

Chen, Kevin; Ou, Xiao-Ming; Chen, Gao; Choi, Si Ho; Shih, Jean C

2005-03-25

Monoamine oxidase catalyzes the oxidative deamination of a number of neurotransmitters. A deficiency in monoamine oxidase A results in aggressive behavior in both humans and mice. Studies on the regulation of monoamine oxidase A gene expression have shown that the Sp1 family is important for monoamine oxidase A expression. To search for novel transcription factors, the sequences of three Sp1 sites in the monoamine oxidase A core promoter were used in the yeast one-hybrid system to screen a human cDNA library. A novel repressor, R1 (RAM2), has been cloned. The R1 cDNA encodes a protein with 454 amino acids and an open reading frame at the 5'-end. The transfection of R1 in a human neuroblastoma cell line, SK-N-BE (2)-C, inhibited the monoamine oxidase A promoter and enzymatic activity. The degree of inhibition of monoamine oxidase A by R1 correlated with the level of R1 protein expression. R1 was also found to repress monoamine oxidase A promoter activity within a natural chromatin environment. A gel-shift assay indicated that the endogenous R1 protein in SK-N-BE (2)-C cells interacted with the R1 binding sequence. R1 also bound directly to the natural monoamine oxidase A promoter in vivo as shown by chromatin immunoprecipitation assay. Immunocytochemical analysis showed that R1 was expressed in both cytosol and nucleus, which suggested a role for R1 in transcriptional regulation. Northern blot analysis revealed the presence of endogenous R1 mRNA in human brain and peripheral tissues. Taken together, this study shows that R1 is a novel repressor that inhibits monoamine oxidase A gene expression.

Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.

PubMed

Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A; Esko, Jeffrey D; Linhardt, Robert J; Sharfstein, Susan T

2012-03-01

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary. Copyright © 2012 Elsevier Inc. All rights reserved.

Wnt signaling inhibits cementoblast differentiation and promotes proliferation.

PubMed

Nemoto, Eiji; Koshikawa, Yohei; Kanaya, Sousuke; Tsuchiya, Masahiro; Tamura, Masato; Somerman, Martha J; Shimauchi, Hidetoshi

2009-05-01

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3beta, increased beta-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras

PubMed Central

Acin, Sergio; Li, Zhongyou; Mejia, Olga; Roop, Dennis R; El-Naggar, Adel K; Caulin, Carlos

2015-01-01

Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53R172H, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53R172H. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53R172H. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53. PMID:21952947

BRCA1 is expressed in uterine serous carcinoma (USC) and controls insulin-like growth factor I receptor (IGF-IR) gene expression in USC cell lines.

PubMed

Amichay, Keren; Kidron, Debora; Attias-Geva, Zohar; Schayek, Hagit; Sarfstein, Rive; Fishman, Ami; Werner, Haim; Bruchim, Ilan

2012-06-01

The insulin-like growth factor I receptor (IGF-IR) and BRCA1 affect cell growth and apoptosis. Little information is available about BRCA1 activity on the IGF signaling pathway. This study evaluated the effect of BRCA1 on IGF-IR expression. BRCA1 and IGF-IR immunohistochemistry on archival tissues (35 uterine serous carcinomas [USCs] and 17 metastases) were performed. USPC1 and USPC2 cell lines were transiently cotransfected with an IGF-IR promoter construct driving a luciferase reporter gene and a BRCA1 expression plasmid. Endogenous IGF-IR levels were evaluated by Western immunoblotting. We found high BRCA1 and IGF-IR protein expression in primary and metastatic USC tumors. All samples were immunostained for BRCA1-71% strongly stained; and 33/35 (94%) were stained positive for IGF-IR-2 (6%) strongly stained. No difference in BRCA1 and IGF-IR staining intensity was noted between BRCA1/2 mutation carriers and noncarriers. Metastatic tumors stained more intensely for BRCA1 than did the primary tumor site (P = 0.041) and with borderline significance for IGF-IR (P = 0.069). BRCA1 and IGF-IR staining did not correlate to survival. BRCA1 expression led to 35% and 54% reduction in IGF-IR promoter activity in the USPC1 and USCP2 cell lines, respectively. Western immunoblotting showed a decline in phosphorylated IGF-IR and phosphorylated AKT in both transiently and stably transfected cells. BRCA1 and IGF-IR are highly expressed in USC tumors. BRCA1 suppresses IGF-IR gene expression and activity. These findings suggest a possible biological link between the BRCA1 and the IGF-I signaling pathways in USC. The clinical implications of this association need to be explored.

Endogenous small RNAs and antibacterial immunity in plants.

PubMed

Jin, Hailing

2008-08-06

Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virus-derived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity.

When politics froze fashion: the effect of the Cultural Revolution on naming in Beijing.

PubMed

Obukhova, Elena; Zuckerman, Ezra W; Zhang, Jiayin

2014-09-01

The authors examine the popularity of boys' given names in Beijing before and after the onset of the Cultural Revolution to clarify how exogenous and endogenous factors interact to shape fashion. Whereas recent work in the sociology of culture emphasizes the importance of endogenous processes in explaining fashion, their analysis demonstrates two ways in which politics shaped cultural expression during the Cultural Revolution: by promoting forms of expression reflecting prevailing political ideology and by limiting individuals' willingness to act differently. As argued by Lieberson and developed further in this article, the second condition is important because endogenous fashion cycles require a critical mass of individuals who seek to differentiate themselves from common practice. Exogenous factors can influence the operation of the endogenous factors. The authors discuss the implications of their study for understanding the nature of conformity under authoritarian regimes and social conditions supporting individual expression.

Comparative analysis of endogenous hormones level in two soybean (Glycine max L.) lines differing in waterlogging tolerance

PubMed Central

Kim, Yoon-Ha; Hwang, Sun-Joo; Waqas, Muhammad; Khan, Abdul L.; Lee, Joon-Hee; Lee, Jeong-Dong; Nguyen, Henry T.; Lee, In-Jung

2015-01-01

Waterlogged condition due to flooding is one of the major abiotic stresses that drastically affect the soybean growth and yield around the world. As a result, many breeders have focused on the development of waterlogging tolerance in soybean varieties, and thus, several tolerant varieties were developed. However, the physiological mechanism of waterlogging tolerance is not yet fully understood. We particularly studied the endogenous hormones regulation during waterlogging in two contrasting soybean genotypes. According to our results, adventitious roots were better developed in the waterlogging tolerant line (WTL) than in the waterlogging susceptible line (WSL). Endogenous hormones also showed significant differences between WTL and WSL. The ethylene production ratio was higher in WTL than in WSL, and methionine was higher in WTL than in WSL. Other endogenous abscisic acid (ABA) contents were lower in WTL than in WSL. Conversely, gibberellic acid (GA) showed a tendency to be high in WTL, especially the levels of the bioactive GA4. The ratio of total GA and ABA was significantly higher in WTL than in WSL. Anatomical study of the root revealed that aerenchyma cells in the stele were better developed in WTL than in WSL. PMID:26442028

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential

PubMed Central

Sonke, Eric; Verrydt, Megan; Postenka, Carl O.; Pardhan, Siddika; Willie, Chantalle J.; Mazzola, Clarisse R.; Hammers, Matthew D.; Pluth, Michael D.; Lobb, Ian; Power, Nicholas E.; Chambers, Ann F.; Leong, Hon S.; Sener, Alp

2016-01-01

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel–Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease. PMID:26068241

Selective Activation of N,N'-Diacyl Rhodamine Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).

PubMed

Abney, Kristopher K; Ramos-Hunter, Susan J; Romaine, Ian M; Godwin, J Shawn; Sulikowski, Gary A; Weaver, Charles David

2018-04-21

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated Porcine Liver Esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel model for development, organization, and function of gonadotropes in fish pituitary.

PubMed

Golan, Matan; Biran, Jakob; Levavi-Sivan, Berta

2014-01-01

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of the reproductive axis in vertebrates. Despite the high popularity of zebrafish as a model organism for studying reproductive functions, to date no transgenic zebrafish with labeled gonadotropes have been introduced. Using gonadotropin regulatory elements from tilapia, we generated two transgenic zebrafish lines with labeled gonadotropes. The tilapia and zebrafish regulatory sequences were highly divergent but several conserved elements allowed the tilapia promoters to correctly drive the transgenes in zebrafish pituitaries. FSH cells reacted to stimulation with gonadotropin releasing hormone by proliferating and showing increased transgene fluorescence, whereas estrogen exposure caused a decrease in cell number and transgene fluorescence. Transgene fluorescence reflected the expression pattern of the endogenous fshb gene. Ontogenetic expression of the transgenes followed typical patterns, with FSH cells appearing early in development, and LH cells appearing later and increasing dramatically in number with the onset of puberty. Our transgenic lines provide a powerful tool for investigating the development, anatomy, and function of the reproductive axis in lower vertebrates.

Ethanol Metabolism by HeLa Cells Transduced with Human Alcohol Dehydrogenase Isoenzymes: Control of the Pathway by Acetaldehyde Concentration†

PubMed Central

Matsumoto, Michinaga; Cyganek, Izabela; Sanghani, Paresh C.; Cho, Won Kyoo; Liangpunsakul, Suthat; Crabb, David W.

2010-01-01

Background Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. Methods The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low Km aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I ADH (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. Results The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs were constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. Conclusion The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady–state acetaldehyde concentration in hepatocytes during ethanol metabolism. PMID:21166830

Flexible tools for gene expression and silencing in tomato.

PubMed

Fernandez, Ana I; Viron, Nicolas; Alhagdow, Moftah; Karimi, Mansour; Jones, Matthew; Amsellem, Ziva; Sicard, Adrien; Czerednik, Anna; Angenent, Gerco; Grierson, Donald; May, Sean; Seymour, Graham; Eshed, Yuval; Lemaire-Chamley, Martine; Rothan, Christophe; Hilson, Pierre

2009-12-01

As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

Axed MUC4 (MUC4/X) aggravates pancreatic malignant phenotype by activating integrin-β1/FAK/ERK pathway.

PubMed

Jahan, Rahat; Macha, Muzafar A; Rachagani, Satyanarayana; Das, Srustidhar; Smith, Lynette M; Kaur, Sukhwinder; Batra, Surinder K

2018-08-01

Alternative splicing is evolving as an eminent player of oncogenic signaling for tumor development and progression. Mucin 4 (MUC4), a type I membrane-bound mucin, is differentially expressed in pancreatic cancer (PC) and plays a critical role in its progression and metastasis. However, the molecular implications of MUC4 splice variants during disease pathogenesis remain obscure. The present study delineates the pathological and molecular significance of a unique splice variant of MUC4, MUC4/X, which lacks the largest exon 2, along with exon 3. Exon 2 encodes for the highly glycosylated tandem repeat (TR) domain of MUC4 and its absence creates MUC4/X, which is devoid of TR. Expression analysis from PC clinical samples revealed significant upregulation of MUC4/X in PC tissues with most differential expression in poorly differentiated tumors. In vitro studies suggest that overexpression of MUC4/X in wild-type-MUC4 (WT-MUC4) null PC cell lines markedly enhanced PC cell proliferation, invasion, and adhesion to extracellular matrix (ECM) proteins. Furthermore, MUC4/X overexpression leads to an increase in the tumorigenic potential of PC cells in orthotopic transplantation studies. In line with these findings, doxycycline-induced expression of MUC4/X in an endogenous WT-MUC4 expressing PC cell line (Capan-1) also displayed enhanced cell proliferation, invasion, and adhesion to ECM, compared to WT-MUC4 alone, emphasizing its direct involvement in the aggressive behavior of PC cells. Investigation into the molecular mechanism suggested that MUC4/X facilitated PC tumorigenesis via integrin-β1/FAK/ERK signaling pathway. Overall, these findings revealed the novel role of MUC4/X in promoting and sustaining the oncogenic features of PC. Copyright © 2018 Elsevier B.V. All rights reserved.

The Synthetic Triterpenoid CDDO-Im Inhibits Fatty Acid Synthase Expression and Has Antiproliferative and Proapoptotic Effects in Human Liposarcoma Cells

PubMed Central

Hughes, David T.; Martel, Peter M.; Kinlaw, William B.; Eisenberg, Burton L.

2013-01-01

Liposarcomas constitute a rare group of tumors of mesenchymal origin that are often poorly responsive to therapy. This study characterizes a novel human liposarcoma cell line (LiSa-2) and defines the mechanism of its response to a synthetic triterpenoid. Fatty acid synthase (FAS) is a key enzyme of de-novo fatty acid synthesis and is highly expressed in both human liposarcoma tissue specimens and LiSa-2 cells. Treatment of the LiSa-2 cell line with the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic imidazolide (CDDO-Im) markedly inhibited FAS mRNA expression, FAS protein production and FAS gene promoter activity. As expected, fatty acid synthesis was down regulated, but there was no effect on cellular fatty acid uptake or glycerol-3-phosphate synthesis suggesting a selective inhibition of endogenous fatty acid synthesis. Importantly, CDDO-Im produced a dose-dependent apoptotic effect in the LiSa-2 cell line, and simultaneous treatment with CDDO-Im and the fatty acid synthase inhibitor Cerulenin produced a synergistic cytotoxic effect. Thus, CDDO-Im and Cerulenin act at different loci to inhibit long chain fatty acid synthesis in liposarcoma cells. This study’s demonstration of CDDO-Im inhibition of FAS and Spot 14 (S14) expression is the first report of triterpenoid compounds affecting the fatty acid synthesis pathway. The observed dependence of liposarcomas on lipogenesis to support their growth and survival provides a novel approach to the treatment of liposarcomas with agents that target fatty acid production. PMID:18259941

Expression of apoplast-targeted plant defensin MtDef4.2 confers resistance to leaf rust pathogen Puccinia triticina but does not affect mycorrhizal symbiosis in transgenic wheat.

PubMed

Kaur, Jagdeep; Fellers, John; Adholeya, Alok; Velivelli, Siva L S; El-Mounadi, Kaoutar; Nersesian, Natalya; Clemente, Thomas; Shah, Dilip

2017-02-01

Rust fungi of the order Pucciniales are destructive pathogens of wheat worldwide. Leaf rust caused by the obligate, biotrophic basidiomycete fungus Puccinia triticina (Pt) is an economically important disease capable of causing up to 50 % yield losses. Historically, resistant wheat cultivars have been used to control leaf rust, but genetic resistance is ephemeral and breaks down with the emergence of new virulent Pt races. There is a need to develop alternative measures for control of leaf rust in wheat. Development of transgenic wheat expressing an antifungal defensin offers a promising approach to complement the endogenous resistance genes within the wheat germplasm for durable resistance to Pt. To that end, two different wheat genotypes, Bobwhite and Xin Chun 9 were transformed with a chimeric gene encoding an apoplast-targeted antifungal plant defensin MtDEF4.2 from Medicago truncatula. Transgenic lines from four independent events were further characterized. Homozygous transgenic wheat lines expressing MtDEF4.2 displayed resistance to Pt race MCPSS relative to the non-transgenic controls in growth chamber bioassays. Histopathological analysis suggested the presence of both pre- and posthaustorial resistance to leaf rust in these transgenic lines. MtDEF4.2 did not, however, affect the root colonization of a beneficial arbuscular mycorrhizal fungus Rhizophagus irregularis. This study demonstrates that the expression of apoplast-targeted plant defensin MtDEF4.2 can provide substantial resistance to an economically important leaf rust disease in transgenic wheat without negatively impacting its symbiotic relationship with the beneficial mycorrhizal fungus.

Fourth-Generation Progestins Inhibit 3β-Hydroxysteroid Dehydrogenase Type 2 and Modulate the Biosynthesis of Endogenous Steroids

PubMed Central

Louw-du Toit, Renate; Perkins, Meghan S.; Snoep, Jacky L.; Storbeck, Karl-Heinz; Africander, Donita

2016-01-01

Progestins used in contraception and hormone replacement therapy are synthetic compounds designed to mimic the actions of the natural hormone progesterone and are classed into four consecutive generations. The biological actions of progestins are primarily determined by their interactions with steroid receptors, and factors such as metabolism, pharmacokinetics, bioavailability and the regulation of endogenous steroid hormone biosynthesis are often overlooked. Although some studies have investigated the effects of select progestins on a few steroidogenic enzymes, studies comparing the effects of progestins from different generations are lacking. This study therefore explored the putative modulatory effects of progestins on de novo steroid synthesis in the adrenal by comparing the effects of select progestins from the respective generations, on endogenous steroid hormone production by the H295R human adrenocortical carcinoma cell line. Ultra-performance liquid chromatography/tandem mass spectrometry analysis showed that the fourth-generation progestins, nestorone (NES), nomegestrol acetate (NoMAC) and drospirenone (DRSP), unlike the progestins selected from the first three generations, modulate the biosynthesis of several endogenous steroids. Subsequent assays performed in COS-1 cells expressing human 3βHSD2, suggest that these progestins modulate the biosynthesis of steroid hormones by inhibiting the activity of 3βHSD2. The Ki values determined for the inhibition of human 3βHSD2 by NES (9.5 ± 0.96 nM), NoMAC (29 ± 7.1 nM) and DRSP (232 ± 38 nM) were within the reported concentration ranges for the contraceptive use of these progestins in vivo. Taken together, our results suggest that newer, fourth-generation progestins may exert both positive and negative physiological effects via the modulation of endogenous steroid hormone biosynthesis. PMID:27706226

MicroRNA-100 regulates pancreatic cancer cells growth and sensitivity to chemotherapy through targeting FGFR3.

PubMed

Li, Zhipeng; Li, Xu; Yu, Chao; Wang, Min; Peng, Feng; Xiao, Jie; Tian, Rui; Jiang, Jianxin; Sun, Chengyi

2014-12-01

We intended to investigate the role of microRNA 100 (miR-100) in regulating pancreatic cancer cells' growth in vitro and tumor development in vivo. QTR-PCR was used to examine the expression of miR-100 in pancreatic cancer cell lines and tumor cells from human patients. Lentivirual vector containing miR-100 mimics (lv-miR-100) was used to overexpress miR-100 in MIA PaCa-2 and FCPAC-1 cells. The effects of overexpressing miR-100 on pancreatic cancer cell proliferation and chemosensitivity to cisplatin were examined by cell proliferation essay in vitro. MIA PaCa-2 cells with endogenously overexpressed miR-100 were transplanted into null mice to examine tumor growth in vivo. The predicted target of miR-100, fibroblast growth factor receptor 3 (FGFR3), was downregulated by siRNA to examine its effect on pancreatic cancer cells. We found miR-100 was markedly underexpressed in both pancreatic cancer cell lines and tumor cells from patients. In cancer cells, transfection of lv-miR-100 was able to upregulate endogenous expression of miR-100, inhibited cancer cell proliferation, and increased sensitivities to cisplatin. Overexpressing miR-100 led to significant inhibition on tumor formation in vivo. Luciferase essay showed FGFR3 was direct target of miR-100. FGFR3 was significantly downregulated by overexpressing miR-100 in pancreatic cancer cells and knocking down FGFR3 by siRNA exerted similar effect as miR-100. Our study demonstrated that miR-100 played an important role in pancreatic cancer development, possibly through targeting FGFR3. It may become a new therapeutic target for gene therapy in patients suffered from pancreatic cancer.

microRNA-137 modulates pancreatic cancer cells tumor growth, invasion and sensitivity to chemotherapy

PubMed Central

Xiao, Jie; Peng, Feng; Yu, Chao; Wang, Min; Li, Xu; Li, Zhipeng; Jiang, Jianxin; Sun, Chengyi

2014-01-01

Background: We intended to investigate the role of microRNA 137 (miR-137) in regulating pancreatic cancer cells’ growth in vitro and tumor development in vivo. Methods: QTR-PCR was used to examine the expression of miR-137 in pancreatic cancer cell lines and tumor cells from human patients. Lentivirual vector containing miR-137 mimic was used to overexpress miR-137 in PANC-1 and MIA PaCa-2 cells. The effects of overexpressing miR-137 on pancreatic cancer cell invasion and chemo-sensitivity to 5-fluorouracil (5-FU) were examined by cell migration and survival essays in vitro. The molecular target of miR-137, pleiotropic growth factor (PTN), was down-regulated by siRNA to examine its effects on cancer cell invasion. MIA PaCa-2 cells with endogenously overexpressed miR-137 were transplanted into null mice to examine tumor growth in vivo. Results: We found miR-137 was markedly underexpressed in both pancreatic cancer cell lines and tumor cells from patients. In cancer cells, transfection of lentivirus containing miR-137 mimic was able to markedly upregulate endogenous expression of miR-137, inhibited cancer cell invasion and increased sensitivities to chemotherapy reagent 5-FU. PTN was significantly down-regulated by overexpressing miR-137 in pancreatic cancer cells, and knocking down PTN was effective to rescue the reduced cancer cell invasion ability caused by miR-137 overexpression. More importantly, overexpressing miR-137 led to significant inhibition on tumor formation, including reductions in tumor weight and tumor size in vivo. Conclusion: Our study demonstrated that miR-137 played an important role in pancreatic cancer development. It may become a new therapeutic target for gene therapy in patients suffered from pancreatic cancer. PMID:25550779

Fibre elongation requires normal redox homeostasis modulated by cytosolic ascorbate peroxidase in cotton (Gossypium hirsutum).

PubMed

Guo, Kai; Du, Xueqiong; Tu, Lili; Tang, Wenxin; Wang, Pengcheng; Wang, Maojun; Liu, Zhen; Zhang, Xianlong

2016-05-01

High-quality cotton fibre equates to a more comfortable textile. Fibre length is an important index of fibre quality. Hydrogen peroxide (H2O2) acts as a signalling molecule in the regulation of fibre elongation. Results from in vitro ovule culture suggest that the alteration of fibre cell H2O2 levels affects fibre development. Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS) scavenging enzyme, and we found that GhAPX1AT/DT encoded one member of the previously unrealized group of cytosolic APXs (cAPXs) that were preferentially expressed during the fibre elongation stage. Transgenic cottons with up- and down-regulation of GhAPX1AT/DT were generated to control fibre endogenous levels of H2O2 Suppression of all cAPX (IAO) resulted in a 3.5-fold increase in H2O2 level in fibres and oxidative stress, which significantly suppressed fibre elongation. The fibre length of transgenic lines with over-expression or specific down-regulation of GhAPX1AT/DT did not show any obvious change. However, the fibres in the over-expression lines exhibited higher tolerance to oxidative stress. Differentially expressed genes (DEGs) in fibres at 10 days post-anthesis (DPA) of IAO lines identified by RNA-seq were related to redox homeostasis, signalling pathways, stress responses and cell wall synthesis, and the DEGs that were up-regulated in IAO lines were also up-regulated in the 10 DPA and 20 DPA fibres of wild cotton compared with domesticated cotton. These results suggest that optimal H2O2 levels and redox state regulated by cytosolic APX are key mechanisms regulating fibre elongation, and dysregulation of the increase in H2O2 induces oxidative stress and results in shorter fibres by initiating secondary cell wall-related gene expression. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Aromatase expression in a human osteoblastic cell line increases in response to prostaglandin E(2) in a dexamethasone-dependent fashion.

PubMed

Watanabe, M; Noda, M; Nakajin, S

2007-09-01

Recent progress supports the importance of local estrogen secretion in human bone tissue to increase and maintain bone-mineral density. In a previous report, we found that forskolin (FSK) synergistically induces aromatase (CYP19: a rate-limiting enzyme for estrogen synthesis) expression in dexamethasone (Dex) dependent manner in a human osteoblastic cell line, SV-HFO [Watanabe M, Ohno S, Nakajin S. Forskolin and dexamethasone synergistically induce aromatase (CYP19) expression in the human osteoblastic cell line SV-HFO. Eur J Endocrinol 2005;152:619-24]. In this report, we investigated whether prostaglandin (PG) E(2) induces estrogen production, in other words, if PGE(2) exerts the same effect as FSK because PGE(2) is the major prostanoid in the bone and is one of the key molecules in the osteoblast. We found PGE(2) up-regulates aromatase activity synergistically, but this up-regulation depends on Dex. CYP19 gene expression was also increased synergistically by Dex and PGE(2). Promoter I.4 was activated synergistically by PGE(2) and Dex. PGE(2) receptor, EP(1), EP(2) and EP(4) were involved in the up-regulation of aromatase activity in response to PGE(2) in a Dex-dependent manner. The cAMP-PKA pathway and Ca(2+) signaling pathway were involved in the up-regulation of aromatase activity in response to PGE(2). Furthermore, glucocorticoid response element on promoter I.4 sequence was an essential minimum requirement for its activity and synergism of PGE(2) and Dex. These findings are the first report on osteoblastic cell line which uses predominantly promoter I.4 to drive aromatase expression. These findings also suggest that endogenous PGE(2) produced in bone mainly may synergistically support local estrogen production in osteoblastic cells in the presence of glucocorticoid.

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

PubMed Central

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

2014-01-01

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

Identification of Mouse Serum miRNA Endogenous References by Global Gene Expression Profiles

PubMed Central

Mi, Qing-Sheng; Weiland, Matthew; Qi, Rui-Qun; Gao, Xing-Hua; Poisson, Laila M.; Zhou, Li

2012-01-01

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs and can serve as serum biomarkers for disease diagnosis and prognoses. Lack of reliable serum miRNA endogenous references for normalization in miRNA gene expression makes single miRNA assays inaccurate. Using TaqMan® real-time PCR miRNA arrays with a global gene expression normalization strategy, we have analyzed serum miRNA expression profiles of 20 female mice of NOD/ShiLtJ (n = 8), NOR/LtJ (n = 6), and C57BL/6J (n = 6) at different ages and disease conditions. We identified five miRNAs, miR-146a, miR-16, miR-195, miR-30e and miR-744, to be stably expressed in all strains, which could serve as mouse serum miRNA endogenous references for single assay experiments. PMID:22348064

MicroRNA-300 inhibited glioblastoma progression through ROCK1.

PubMed

Zhou, Fucheng; Li, Yang; Hao, Zhen; Liu, Xuanxi; Chen, Liang; Cao, Yu; Liang, Zuobin; Yuan, Fei; Liu, Jie; Wang, Jianjiao; Zheng, Yongri; Dong, Deli; Bian, Shan; Yang, Baofeng; Jiang, Chuanlu; Li, Qingsong

2016-06-14

Glioblastoma is a common type of brain aggressive tumors and has a poor prognosis. MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAs that play crucial roles in cell proliferation, survival and invasion. Deregulated expression of miR-300 has been studied in a lot of cancers. However, the role of miR-300 in glioblastoma is still unknown. In this study, we demonstrated that miR-300 expression was downregulated in glioblastoma tissues compared with the normal tissues. Lower expression level of miR-300 was observed in thirty cases (75 %, 30/40) of glioblastoma samples compared with the normal samples. Moreover, the overall survival of glioblastoma patients with lower miR-300 expression level was shorter than those with higher miR-300 expression level. In addition, miR-300 expression was also downregulated in glioblastoma cell lines. Overexpression of miR-300 inhibited cell proliferation, cell cycle and invasion in glioblastoma cell line U87 and U251. Moreover, we identified ROCK1 as a direct target of miR-300 in U87 and U251 cells. Overexpression of ROCK1 partially rescued the miR-300-mediated cell growth. ROCK1 expression levels in glioblastoma tissues were higher than that in normal tissues. ROCK1 expression levels were higher in thirty-one cases of glioblastoma samples than their normal samples. Furthermore, the expression level ROCK1 was inversely correlated with the expression level of miR-300. Importantly, overexpression of miR-300 suppressed glioblastoma progression in an established xenograft model. In conclusion, we revealed that miR-300 might act as a tumor suppressor gene through inhibiting ROCK1 in glioblastoma.

MicroRNA-300 inhibited glioblastoma progression through ROCK1

PubMed Central

Hao, Zhen; Liu, Xuanxi; Chen, Liang; Cao, Yu; Liang, Zuobin; Yuan, Fei; Liu, Jie; Wang, Jianjiao; Zheng, Yongri; Dong, Deli; Bian, Shan; Yang, Baofeng; Jiang, Chuanlu; Li, Qingsong

2016-01-01

Glioblastoma is a common type of brain aggressive tumors and has a poor prognosis. MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAs that play crucial roles in cell proliferation, survival and invasion. Deregulated expression of miR-300 has been studied in a lot of cancers. However, the role of miR-300 in glioblastoma is still unknown. In this study, we demonstrated that miR-300 expression was downregulated in glioblastoma tissues compared with the normal tissues. Lower expression level of miR-300 was observed in thirty cases (75 %, 30/40) of glioblastoma samples compared with the normal samples. Moreover, the overall survival of glioblastoma patients with lower miR-300 expression level was shorter than those with higher miR-300 expression level. In addition, miR-300 expression was also downregulated in glioblastoma cell lines. Overexpression of miR-300 inhibited cell proliferation, cell cycle and invasion in glioblastoma cell line U87 and U251. Moreover, we identified ROCK1 as a direct target of miR-300 in U87 and U251 cells. Overexpression of ROCK1 partially rescued the miR-300-mediated cell growth. ROCK1 expression levels in glioblastoma tissues were higher than that in normal tissues. ROCK1 expression levels were higher in thirty-one cases of glioblastoma samples than their normal samples. Furthermore, the expression level ROCK1 was inversely correlated with the expression level of miR-300. Importantly, overexpression of miR-300 suppressed glioblastoma progression in an established xenograft model. In conclusion, we revealed that miR-300 might act as a tumor suppressor gene through inhibiting ROCK1 in glioblastoma. PMID:27145462

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

PubMed Central

2010-01-01

Background Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken. Results We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines. Conclusions From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species. PMID:20184756

Distribution of CaMKIIα expression in the brain in vivo, studied by CaMKIIα-GFP mice

PubMed Central

Wang, Xinjun; Zhang, Chunzhao; Szábo, Gábor; Sun, Qian-Quan

2013-01-01

To facilitate the study of the CaMKIIα function in vivo, a CaMKIIα-GFP transgenic mouse line was generated. Here, our goal is to provide the first neuroanatomical characterization of GFP expression in the CNS of this line of mouse. Overall, CaMKIIα -GFP expression is strong and highly heterogeneous, with the dentate gyrus of the hippocampus as the most abundantly expressed region. In the hippocampus, around 70% of granule and pyramidal neurons expressed strong GFP. In the neocortex, presumed pyramidal neurons were GFP positive: around 32% of layer II/III and 35% of layer VI neurons expressed GFP, and a lower expression rate was found in other layers. In the thalamus and hypothalamus, strong GFP signals were detected in the neuropil. GFP-positive cells were also found in many other regions such as the spinal trigeminal nucleus, cerebellum and basal ganglia. We further compared the GFP expression with specific antibody staining for CaMKIIα and GABA. We found that GFP+ neurons were mostly positive for CaMKIIα-IR throughout the brain, with some exceptions throughout the brain, especially in the deeper layers of neocortex. GFP and GABA-IR marked distinct neuronal populations in most brain regions with the exception of granule cells in the olfactory bulb, purkinje cells in the cerebellar, and some layer I cells in neocortex. In conclusion, GFP expression in the CaMKIIα-GFP mice is similar to the endogenous expression of CaMKIIα protein, thus these mice can be used in in vivo and in vitro physiological studies in which visualization of CaMKIIα- neuronal populations is required. PMID:23632380

A novel microRNA located in the TrkC gene regulates the Wnt signaling pathway and is differentially expressed in colorectal cancer specimens

PubMed Central

Dokanehiifard, Sadat; Yasari, Atena; Najafi, Hadi; Jafarzadeh, Meisam; Nikkhah, Maryam; Mowla, Seyed Javad; Soltani, Bahram M.

2017-01-01

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis, differentiation, and tumorigenesis. TrkC diverse functions might be attributed to the hypothetical non-coding RNAs embedded within the gene. Using bioinformatics approaches, a novel microRNA named TrkC-miR2 was predicted within the TrkC gene capable of regulating the Wnt pathway. For experimental verification of this microRNA, the predicted TrkC-premir2 sequence was overexpressed in SW480 cells, which led to the detection of two mature TrkC-miR2 isomiRs, and their endogenous forms were detected in human cell lines as well. Later, an independent promoter was deduced for TrkC-miR2 after the treatment of HCT116 cells with 5-azacytidine, which resulted in differential expression of TrkC-miR2 and TrkC host gene. RT-quantitative PCR and luciferase assays indicated that the APC2 gene is targeted by TrkC-miR2, and Wnt signaling is up-regulated. Also, Wnt inhibition by using small molecules along with TrkC-miR2 overexpression and TOP/FOP flash assays confirmed the positive effect of TrkC-miR2 on the Wnt pathway. Consistently, TrkC-miR2 overexpression promoted SW480 cell survival, which was detected by flow cytometry, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, and crystal violate analysis. RT-qPCR analysis revealed that TrkC-miR2 is significantly up-regulated (∼70 times) in colorectal tumor tissues compared with their normal pairs. Moreover, the TrkC-miR2 expression level discriminated grades of tumor malignancies, which was consistent with its endogenous levels in HCT116, HT29, and SW480 colorectal cancer cell lines. Finally, an opposite expression pattern was observed for TrkC-miR2 and the APC2 gene in colorectal cancer specimens. In conclusion, here we introduce TrkC-miR2 as a novel regulator of Wnt signaling, which might be a candidate oncogenic colorectal cancer biomarker. PMID:28100780

Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

PubMed Central

Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

2002-01-01

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID:11772392

Effect of caffeine on induction of endogenous type C virus in mouse cells in vitro

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

Niwa, O.; Sugahara, T.

1981-08-01

The effect of caffeine on the expression of murine endogenous virus in mouse cells induced by radiation and chemicals was studied. Postirradiation treatment of K-BALB cells with caffeine enhanced cell killing as well as the induction of xenotropic virus after ultraviolet light irradiation. The degree of enhancement for the virus induction was comparable to that for cell killing. On the other hand, colony-forming ability and the expression of xenotropic virus of K-BALB cells after X-irradiation were unaffected by caffeine. These data suggest a linear relationship between the degree of endogenous virus expression and the amount of lethal damages after irradiation.more » For induction by halogenated pyrimidines, a 24-hr incubation of AKR2B cells with caffeine after 5-iodo-2'-deoxyuridine treatment resulted in marked suppression of the expression of ecotropic virus. On the contrary, in K-BALB cells, caffeine exerted only a small effect on 5-iodo-2'-deoxyuridine-induced expression of ecotropic and xenotropic viruses. These results indicate that, although using the same inducing agent, the pathway of endogenous virus induction may be different for AKR2B cells and for K-BALB cells.« less

The progestational and androgenic properties of medroxyprogesterone acetate: gene regulatory overlap with dihydrotestosterone in breast cancer cells

PubMed Central

Ghatge, Radhika P; Jacobsen, Britta M; Schittone, Stephanie A; Horwitz, Kathryn B

2005-01-01

Introduction Medroxyprogesterone acetate (MPA), the major progestin used for oral contraception and hormone replacement therapy, has been implicated in increased breast cancer risk. Is this risk due to its progestational or androgenic properties? To address this, we assessed the transcriptional effects of MPA as compared with those of progesterone and dihydrotestosterone (DHT) in human breast cancer cells. Method A new progesterone receptor-negative, androgen receptor-positive human breast cancer cell line, designated Y-AR, was engineered and characterized. Transcription assays using a synthetic promoter/reporter construct, as well as endogenous gene expression profiling comparing progesterone, MPA and DHT, were performed in cells either lacking or containing progesterone receptor and/or androgen receptor. Results In progesterone receptor-positive cells, MPA was found to be an effective progestin through both progesterone receptor isoforms in transient transcription assays. Interestingly, DHT signaled through progesterone receptor type B. Expression profiling of endogenous progesterone receptor-regulated genes comparing progesterone and MPA suggested that although MPA may be a somewhat more potent progestin than progesterone, it is qualitatively similar to progesterone. To address effects of MPA through androgen receptor, expression profiling was performed comparing progesterone, MPA and DHT using Y-AR cells. These studies showed extensive gene regulatory overlap between DHT and MPA through androgen receptor and none with progesterone. Interestingly, there was no difference between pharmacological MPA and physiological MPA, suggesting that high-dose therapeutic MPA may be superfluous. Conclusion Our comparison of the gene regulatory profiles of MPA and progesterone suggests that, for physiologic hormone replacement therapy, the actions of MPA do not mimic those of endogenous progesterone alone. Clinically, the complex pharmacology of MPA not only influences its side-effect profile; but it is also possible that the increased breast cancer risk and/or the therapeutic efficacy of MPA in cancer treatment is in part mediated by androgen receptor. PMID:16457685

Identification of suitable reference genes for hepatic microRNA quantitation.

PubMed

Lamba, Vishal; Ghodke-Puranik, Yogita; Guan, Weihua; Lamba, Jatinder K

2014-03-07

MicroRNAs (miRNAs) are short (~22 nt) endogenous RNAs that play important roles in regulating expression of a wide variety of genes involved in different cellular processes. Alterations in microRNA expression patterns have been associated with a number of human diseases. Accurate quantitation of microRNA levels is important for their use as biomarkers and in determining their functions. Real time PCR is the gold standard and the most frequently used technique for miRNA quantitation. Real time PCR data analysis includes normalizing the amplification data to suitable endogenous control/s to ensure that microRNA quantitation is not affected by the variability that is potentially introduced at different experimental steps. U6 (RNU6A) and RNU6B are two commonly used endogenous controls in microRNA quantitation. The present study was designed to investigate inter-individual variability and gender differences in hepatic microRNA expression as well as to identify the best endogenous control/s that could be used for normalization of real-time expression data in liver samples. We used Taqman based real time PCR to quantitate hepatic expression levels of 22 microRNAs along with U6 and RNU6B in 50 human livers samples (25 M, 25 F). To identify the best endogenous controls for use in data analysis, we evaluated the amplified candidates for their stability (least variability) in expression using two commonly used software programs: Normfinder and GeNormplus, Both Normfinder and GeNormplus identified U6 to be among the least stable of all the candidates analyzed, and RNU6B was also not among the top genes in stability. mir-152 and mir-23b were identified to be the two most stable candidates by both Normfinder and GeNormplus in our analysis, and were used as endogenous controls for normalization of hepatic miRNA levels. Measurements of microRNA stability indicate that U6 and RNU6B are not suitable for use as endogenous controls for normalizing microRNA relative quantitation data in hepatic tissue, and their use can led to possibly erroneous conclusions.

Hydroquinone induces DNA hypomethylation-independent overexpression of retroelements in human leukemia and hematopoietic stem cells.

PubMed

Conti, Anastasia; Rota, Federica; Ragni, Enrico; Favero, Chiara; Motta, Valeria; Lazzari, Lorenza; Bollati, Valentina; Fustinoni, Silvia; Dieci, Giorgio

2016-06-10

Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous leukemia risk. Although altered DNA methylation has been reported in both benzene-exposed human subjects and HQ-exposed cultured cells, the inventory of benzene metabolite effects on the epigenome is only starting to be established. In this study, we used a monocytic leukemia cell line (THP-1) and hematopoietic stem cells (HSCs) from cord blood to investigate the effects of HQ treatment on the expression of the three most important families of retrotransposons in the human genome: LINE-1, Alu and Endogenous retroviruses (HERVs), that are normally subjected to tight epigenetic silencing. We found a clear tendency towards increased retrotransposon expression in response to HQ exposure, more pronounced in the case of LINE-1 and HERV. Such a partial loss of silencing, however, was generally not associated with HQ-induced DNA hypomethylation. On the other hand, retroelement derepression was also observed in the same cells in response to the hypomethylating agent decitabine. These observations suggest the existence of different types of epigenetic switches operating at human retroelements, and point to retroelement activation in response to benzene-derived metabolites as a novel factor deserving attention in benzene carcinogenesis studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway

PubMed Central

Kasper, Grit; Reule, Matthias; Tschirschmann, Miriam; Dankert, Niels; Stout-Weider, Karen; Lauster, Roland; Schrock, Evelin; Mennerich, Detlev; Duda, Georg N; Lehmann, Kerstin E

2007-01-01

Background Stromelysin-3 (ST-3) is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized. Methods The present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed. Results Using both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts. Conclusion These data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour fibroblasts leads to the stimulation of the IGF-1R pathway in carcinoma cells, thus enhancing their proliferative capacity. In addition, further different cellular processes seem to be activated by ST-3, possibly accounting for the dual role of ST-3 in tumour progression and metastasis. PMID:17233884

Transcriptional inhibition of p21{sup WAF1/CIP1} gene (CDKN1) expression by survivin is at least partially p53-dependent: Evidence for survivin acting as a transcription factor or co-factor

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

Tang, Lei; Pre-Doctoral Chinese Fellowship Student, Second West China Hospital, Sichuan University, Sichuan; Ling, Xiang

2012-05-04

Highlights: Black-Right-Pointing-Pointer Survivin inhibits the expression of p21 protein, mRNA and promoter activity. Black-Right-Pointing-Pointer Survivin neutralizes p53-induced p21 expression and promoter activity. Black-Right-Pointing-Pointer Survivin physically interacts with p53 in cancer cells. Black-Right-Pointing-Pointer Genetic silencing of endogenous survivin upregulates p21 in p53 wild type cancer cells. Black-Right-Pointing-Pointer Both p53 and survivin interacts on the two p53-binding sites in the p21 promoter. -- Abstract: Growing evidence suggests a role for the antiapoptotic protein survivin in promotion of cancer cell G1/S transition and proliferation. However, the underlying mechanism is unclear. Further, although upregulation of p21{sup WAF1/CIP1} by p53 plays an important role inmore » p53-mediated cell G1 arrests in response to various distresses, it is unknown whether survivin plays a role in the regulation of p21{sup WAF1/CIP1} expression. Here, we report that exogenous expression of survivin in p53-wild type MCF-7 breast cancer cells inhibits the expression of p21{sup WAF1/CIP1} protein, mRNA and promoter activity, while the survivin C84A mutant and antisense failed to do so. Cotransfection experiments in the p53 mutant H1650 lung cancer cell line showed that survivin neutralizes p53-induced p21{sup WAF1/CIP1} expression and promoter activity. Importantly, genetically silencing of endogenous survivin using lentiviral survivin shRNA also enhances endogenous p21 in p53 wild type cancer cells, suggesting the physiological relevance of the fining. We further demonstrated that both p53 and survivin interacts on the two p53-binding sites in the p21{sup WAF1/CIP1} promoter (-2313 to -2212; -1452 to -1310), and survivin physically interacts with p53 in cancer cells. Together, we propose that survivin may act as a transcription factor or cofactor to interact with p53 on the p21{sup WAF1/CIP1} promoter leading to the inhibition of p21{sup WAF1/CIP1} expression at least in part by neutralizing p53-mediated transcriptional activation of the p21 gene.« less

1α,25(OH)2D3 differentially regulates miRNA expression in human bladder cancer cells.

PubMed

Ma, Yingyu; Hu, Qiang; Luo, Wei; Pratt, Rachel N; Glenn, Sean T; Liu, Song; Trump, Donald L; Johnson, Candace S

2015-04-01

Bladder cancer is the fourth most commonly diagnosed cancer in men and eighth leading cause of cancer-related death in the US. Epidemiological and experimental studies strongly suggest a role for 1α,25(OH)2D3 in cancer prevention and treatment. The antitumor activities of 1α,25(OH)2D3 are mediated by the induction of cell cycle arrest, apoptosis, differentiation and the inhibition of angiogenesis and metastasis. miRNAs play important regulatory roles in cancer development and progression. However, the role of 1α,25(OH)2D3 in the regulation of miRNA expression and the potential impact in bladder cancer has not been investigated. Therefore, we studied 1α,25(OH)2D3-regulated miRNA expression profiles in human bladder cancer cell line 253J and the highly tumorigenic and metastatic derivative line 253J-BV by miRNA qPCR panels. 253J and 253J-BV cells express endogenous vitamin D receptor (VDR), which can be further induced by 1α,25(OH)2D3. VDR target gene 24-hydroxylase was induced by 1α,25(OH)2D3 in both cell lines, indicating functional 1α,25(OH)2D3 signaling. The miRNA qPCR panel assay results showed that 253J and 253J-BV cells have distinct miRNA expression profiles. Further, 1α,25(OH)2D3 differentially regulated miRNA expression profiles in 253J and 253J-BV cells in a dynamic manner. Pathway analysis of the miRNA target genes revealed distinct patterns of contribution to the molecular functions and biological processes in the two cell lines. In conclusion, 1α,25(OH)2D3 differentially regulates the expression of miRNAs, which may contribute to distinct biological functions, in human bladder 253J and 253J-BV cells. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Copyright © 2014 Elsevier Ltd. All rights reserved.

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

PubMed

Musarò, A; Rosenthal, N

1999-04-01

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

A Malus Crabapple Chalcone Synthase Gene, McCHS, Regulates Red Petal Color and Flavonoid Biosynthesis

PubMed Central

Song, Tingting; Yao, Yuncong

2014-01-01

Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, ‘Royalty’ and ‘Flame’, have dark red and white petals respectively, while the intermediate cultivar ‘Radiant’ has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in ‘Radiant’. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple. PMID:25357207

Parvovirus-derived endogenous viral elements in two South American rodent genomes.

PubMed

Arriagada, Gloria; Gifford, Robert J

2014-10-01

We describe endogenous viral elements (EVEs) derived from parvoviruses (family Parvoviridae) in the genomes of the long-tailed chinchilla (Chinchilla lanigera) and the degu (Octodon degus). The novel EVEs include dependovirus-related elements and representatives of a clearly distinct parvovirus lineage that also has endogenous representatives in marsupial genomes. In the degu, one dependovirus-derived EVE was found to carry an intact reading frame and was differentially expressed in vivo, with increased expression in the liver. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Polycation-induced Cell Membrane Permeability Does Not Enhance Cellular Uptake or Expression Efficiency of Delivered DNA

PubMed Central

Prevette, Lisa E.; Mullen, Douglas G.; Banaszak Holl, Mark M.

2010-01-01

Polycationic materials commonly used to delivery DNA to cells are known to induce cell membrane porosity in a charge-density dependent manner. It has been suggested that these pores may provide a mode of entry of the polymer-DNA complexes (polyplexes) into cells. To examine the correlation between membrane permeability and biological activity, we used two-color flow cytometry on two mammalian cell lines to simultaneously measure gene expression of a plasmid DNA delivered with four common nonviral vectors and cellular uptake of normally excluded fluorescent dye molecules of two different sizes, 668 Da and 2 MDa. We also followed gene expression in cells sorted based on the retention of endogenous fluorescein. We have found that cell membrane porosity caused by polycationic vectors does not enhance internalization or gene expression. Based on this single-cell study, membrane permeability is found to be an unwanted side effect that limits transfection efficiency, possibly through leakage of the delivered nucleic acid through the pores prior to transcription and translation and/or activation of cell defense mechanisms that restrict transgene expression. PMID:20349965

Endogenous and Uric Acid-Induced Activation of NLRP3 Inflammasome in Pregnant Women with Preeclampsia.

PubMed

Matias, Mariana Leticia; Romão, Mariana; Weel, Ingrid Cristina; Ribeiro, Vanessa Rocha; Nunes, Priscila Rezeck; Borges, Vera Therezinha; Araújo, João Pessoa; Peraçoli, José Carlos; de Oliveira, Leandro; Peraçoli, Maria Terezinha

2015-01-01

Preeclampsia (PE) is a specific syndrome of pregnancy, characterized by hypertension and proteinuria. This pathology is associated with hyperuricemia and elevated serum levels of inflammatory cytokines. Uric acid crystals may activate an intracellular complex called inflammasome, which is important for processing and release of inflammatory cytokines. This study investigated the state of monocyte activation, both endogenous and stimulated with monosodium urate (MSU), by gene expression of NLRP1 and NLRP3 receptors as well as their association with inflammatory cytokines expression. Monocytes were obtained from peripheral blood of 23 preeclamptic pregnant women, 23 normotensive pregnant women (NT) and 23 healthy non-pregnant women (NP). Inflammasome activation was evaluated by the gene expression of NLRP1, NLRP3, caspase-1, IL-1β, IL-18 and TNF-α by RT-qPCR in unstimulated monocytes (endogenous expression), or after cell stimulation with MSU (stimulated expression). The concentration of cytokines was assessed by ELISA. In preeclamptic pregnant women, gene expression of NLRP1, NLRP3, caspase-1, IL-1β and TNF-α by monocytes stimulated or not with MSU was significantly higher than in NT and NP groups. Stimulation of monocytes from preeclamptic and non-pregnant women with MSU induced increased gene expression of NLRP3, caspase-1 and TNF-α in relation to the endogenous expression in these groups, while this was not observed in the NT group. The cytokine determination showed that monocytes from women with PE produced higher endogenous levels of IL-1β, IL-18 and TNF-α compared to the other groups, while the stimulus with MSU led to higher production of these cytokines in preeclamptic group than in the NT group. In conclusion, the results showed increased basal gene expression of NLRP1 and NLRP3 receptors in monocytes from PE group. These cells stimulation with MSU demonstrates that uric acid plays a role in NLRP3 inflammasome activation, suggesting the participation of this inflammatory complex in the pathogenesis of preeclampsia.

Tryptophan hydroxylase 2 (TPH2) in a neuronal cell line: modulation by cell differentiation and NRSF/rest activity.

PubMed

Gentile, Maria Teresa; Nawa, Yukino; Lunardi, Gianluigi; Florio, Tullio; Matsui, Hiroaki; Colucci-D'Amato, Luca

2012-12-01

Serotonin (5-HT) is a neurotransmitter involved in many aspects of the neuronal function. The synthesis of 5-HT is initiated by the hydroxylation of tryptophan, catalyzed by tryptophan hydroxylase (TPH). Two isoforms of TPH (TPH1 and TPH2) have been identified, with TPH2 almost exclusively expressed in the brain. Following TPH2 discovery, it was reported that polymorphisms of both gene and non-coding regions are associated with a spectrum of psychiatric disorders. Thus, insights into the mechanisms that specifically regulate TPH2 expression and its modulation by exogenous stimuli may represent a new therapeutic approach to modify serotonergic neurotransmission. To this aim, a CNS-originated cell line expressing TPH2 endogenously represents a valid model system. In this study, we report that TPH2 transcript and protein are modulated by neuronal differentiation in the cell line A1 mes-c-myc (A1). Moreover, we show luciferase activity driven by the human TPH2 promoter region and demonstrate that upon mutation of the NRSF/REST responsive element, the promoter activity strongly increases with cell differentiation. Our data suggest that A1 cells could represent a model system, allowing an insight into the mechanisms of regulation of TPH2 and to identify novel therapeutic targets in the development of drugs for the management of psychiatric disorders. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

LRIG1 modulates aggressiveness of head and neck cancers by regulating EGFR-MAPK-SPHK1 signaling and extracellular matrix remodeling.

PubMed

Sheu, J J-C; Lee, C-C; Hua, C-H; Li, C-I; Lai, M-T; Lee, S-C; Cheng, J; Chen, C-M; Chan, C; Chao, S C-C; Chen, J-Y; Chang, J-Y; Lee, C-H

2014-03-13

EGFR overexpression and chromosome 3p deletion are two frequent events in head and neck cancers. We previously mapped the smallest region of recurrent copy-number loss at 3p12.2-p14.1. LRIG1, a negative regulator of EGFR, was found at 3p14, and its copy-number loss correlated with poor clinical outcome. Inducible expression of LRIG1 in head and neck cancer TW01 cells, a line with low LRIG1 levels, suppressed cell proliferation in vitro and tumor growth in vivo. Gene expression profiling, quantitative RT-PCR, chromatin immunoprecipitation, and western blot analysis demonstrated that LRIG1 modulated extracellular matrix (ECM) remodeling and EGFR-MAPK-SPHK1 transduction pathway by suppressing expression of EGFR ligands/activators, MMPs and SPHK1. In addition, LRIG1 induction triggered cell morphology changes and integrin inactivation, which coupled with reduced SNAI2 expression. By contrast, knockdown of endogenous LRIG1 in TW06 cells, a line with normal LRIG1 levels, significantly enhanced cell proliferation, migration and invasiveness. Such tumor-promoting effects could be abolished by specific MAPK or SPHK1 inhibitors. Our data suggest LRIG1 as a tumor suppressor for head and neck cancers; LRIG1 downregulation in cancer cells enhances EGFR-MAPK-SPHK1 signaling and ECM remodeling activity, leading to malignant phenotypes of head and neck cancers.

Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription

PubMed Central

Faura Tellez, Grissel; Vandepoele, Karl; Brouwer, Uilke; Koning, Henk; Elderman, Robin M.; Hackett, Tillie-Louise; Willemse, Brigitte W. M.; Holloway, John; Van Roy, Frans; Koppelman, Gerard H.

2015-01-01

Genetic studies have identified Protocadherin-1 (PCDH1) and Mothers against decapentaplegic homolog-3 (SMAD3) as susceptibility genes for asthma. PCDH1 is expressed in bronchial epithelial cells and has been found to interact with SMAD3 in yeast two-hybrid (Y2H) overexpression assays. Here, we test whether PCDH1 and SMAD3 interact at endogenous protein levels in bronchial epithelial cells and evaluate the consequences thereof for transforming growth factor-β1 (TGF-β1)-induced gene transcription. We performed Y2H screens and coimmunoprecipitation (co-IP) experiments of PCDH1 and SMAD3 in HEK293T and 16HBE14o− (16HBE) cell lines. Activity of a SMAD3-driven luciferase reporter gene in response to TGF-β1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-β1-induced gene expression was quantified in BEAS-2B clones overexpressing PCDH1 and in human primary bronchial epithelial cells (PBECs) transfected with PCDH1-siRNA. We confirm PCDH1 and SMAD3 interactions by Y2H and by co-IP in HEK293T cells overexpressing both proteins, and at endogenous protein levels in 16HBE cells. TGF-β-induced activation of a SMAD3-driven reporter was reduced by exogenous PCDH1 in BEAS2B cells, whereas it was increased by siRNA-mediated knockdown of endogenous PCDH1 in 16HBE cells. Overexpression of PCDH1 suppressed expression of TGF-β target genes in BEAS-2B cells, whereas knockdown of PCDH1 in human PBECs increased TGF-β-induced gene expression. In conclusion, we demonstrate that PCDH1 binds to SMAD3 and regulates its activation by TGF-β signaling in bronchial epithelial cells. We propose that PCDH1 and SMAD3 act in a single pathway in asthma susceptibility that affects sensitivity of the airway epithelium to TGF-β. PMID:26209277

The Kinetics of G2 and M Transitions Regulated by B Cyclins

PubMed Central

Huang, Yehong; Sramkoski, R. Michael; Jacobberger, James W.

2013-01-01

B cyclins regulate G2-M transition. Because human somatic cells continue to cycle after reduction of cyclin B1 (cycB1) or cyclin B2 (cycB2) by RNA interference (RNAi), and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated HeLa BD™ Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were RNAi resistant. Cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy. This suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFPs did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous cyclin in single kd experiments, and either cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle, while cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet residual levels are sufficient to promote mitosis in the presence of the dispensible cyclin B2. We discuss a simple model that would explain most data if cyclin B1 is necessary. PMID:24324638

High-Content Screening Identifies Src Family Kinases as Potential Regulators of AR-V7 Expression and Androgen-Independent Cell Growth.

PubMed

Szafran, Adam T; Stephan, Cliff; Bolt, Michael; Mancini, Maureen G; Marcelli, Marco; Mancini, Michael A

2017-01-01

AR-V7 is an androgen receptor (AR) splice variant that lacks the ligand-binding domain and is isolated from prostate cancer cell lines. Increased expression of AR-V7 is associated with the transition from hormone-sensitive prostate cancer to more advanced castration-resistant prostate cancer (CRPC). Due to the loss of the ligand-binding domain, AR-V7 is not responsive to traditional AR-targeted therapies, and the mechanisms that regulate AR-V7 are still incompletely understood. Therefore, we aimed to explore existing classes of small molecules that may regulate AR-V7 expression and intracellular localization and their potential therapeutic role in CRPC. We used AR high-content analysis (AR-HCA) to characterize the effects of a focused library of well-characterized clinical compounds on AR-V7 expression at the single-cell level in PC3 prostate cancer cells stably expressing green fluorescent protein (GFP)-AR-V7 (GFP-AR-V7:PC3). In parallel, an orthogonal AR-HCA screen of a small interfering (si)RNA library targeting 635 protein kinases was performed in GFP-AR-V7:PC3. The effect of the Src-Abl inhibitor PD 180970 was further characterized using cell-proliferation assays, quantitative PCR, and western blot analysis in multiple hormone-sensitive and CRPC cell lines. Compounds that tended to target Akt, Abl, and Src family kinases (SFKs) decreased overall AR-V7 expression, nuclear translocation, absolute nuclear level, and/or altered nuclear distribution. We identified 20 protein kinases that, when knocked down, either decreased nuclear GFP-AR-V7 levels or altered AR-V7 nuclear distribution, a set that included the SFKs Src and Fyn. The Src-Abl dual kinase inhibitor PD180970 decreased expression of AR-V7 by greater than 46% and decreased ligand-independent transcription of AR target genes in the 22RV1 human prostate carcinoma cell line. Further, PD180970 inhibited androgen-independent cell proliferation in endogenous-AR-V7-expressing prostate cancer cell lines and also overcame bicalutamide resistance observed in the 22RV1 cell line. SFKs, especially Src and Fyn, may be important upstream regulators of AR-V7 expression and represent promising targets in a subset of CRPCs expressing high levels of AR-V7. Prostate 77:82-93, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A short region of the promoter of the breast cancer associated PLU-1 gene can regulate transcription in vitro and in vivo.

PubMed

Catteau, Aurélie; Rosewell, Ian; Solomon, Ellen; Taylor-Papadimitriou, Joyce

2004-07-01

The recently cloned gene PLU-1 shows restricted expression in adult tissues, with high expression being found in testis, and transiently in the pregnant mammary gland. However, both the gene and the protein product are specifically up-regulated in breast cancer. To investigate the control of expression of the PLU-1 gene, we have cloned and functionally characterised the 5' flanking region of the gene, which was found to contain another putative gene. Two transcription start sites of the PLU-1 gene were mapped by 5' RACE. A short proximal 249 bp region was defined using reporter gene assays, which encompasses the major transcription start site and exhibits a strong constitutive promoter activity in all cell lines tested. However, regions upstream of this sequence repress transcription more effectively in a non-malignant breast cell line as compared to breast cancer cell lines. The 249 bp region is GC-rich and includes consensus Sp1 sites, GC boxes, cAMP-responsive element (CRE) and other putative cis-elements. Mutational analysis showed that two intact conserved Sp1 binding sites (shown here to bind Sp1 and/or Sp3) are critical for constitutive promoter activity, while a negative role for a neighbouring GC box is indicated. The sequence of the core promoter is highly conserved in the mouse and Plu-1 expression in the mouse embryo has been documented. Using transgenesis, we therefore examined the ability of the 249 bp fragment to control expression of a reporter gene during embryogenesis. We found that not only is the core promoter sufficient to activate transcription in vivo, but that the expression of the reporter gene coincides both temporally and spatially with regions where endogenous Plu-1 is highly expressed. This suggests that tissue specific controlling elements are found within the short fragment and are functional in the embryonic environment.

Lysophosphatidic Acid Acyltransferase β (LPAATβ) Promotes the Tumor Growth of Human Osteosarcoma

PubMed Central

Rastegar, Farbod; Gao, Jian-Li; Shenaq, Deana; Luo, Qing; Shi, Qiong; Kim, Stephanie H.; Jiang, Wei; Wagner, Eric R.; Huang, Enyi; Gao, Yanhong; Shen, Jikun; Yang, Ke; He, Bai-Cheng; Chen, Liang; Zuo, Guo-Wei; Luo, Jinyong; Luo, Xiaoji; Bi, Yang; Liu, Xing; Li, Mi; Hu, Ning; Wang, Linyuan; Luther, Gaurav; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan

2010-01-01

Background Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase β (LPAATβ, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATβ can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATβ has been reported in several types of human tumors, the role of LPAATβ in osteosarcoma progression has yet to be elucidated. Methodology/Principal Findings Endogenous expression of LPAATβ in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATβ and silencing LPAATβ expression is employed to determine the effect of LPAATβ on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATβ is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATβ promotes osteosarcoma cell proliferation and migration, while silencing LPAATβ expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATβ effectively promotes tumor growth, while knockdown of LPAATβ expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma. Conclusions/Significance Our results strongly suggest that LPAATβ expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATβ may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATβ may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors. PMID:21152068

Effects of the single and combined treatment with dopamine agonist, somatostatin analog and mTOR inhibitors in a human lung carcinoid cell line: an in vitro study.

PubMed

Pivonello, Claudia; Rousaki, Panagoula; Negri, Mariarosaria; Sarnataro, Maddalena; Napolitano, Maria; Marino, Federica Zito; Patalano, Roberta; De Martino, Maria Cristina; Sciammarella, Concetta; Faggiano, Antongiulio; Rocco, Gaetano; Franco, Renato; Kaltsas, Gregory A; Colao, Annamaria; Pivonello, Rosario

2017-06-01

Somatostatin analogues and mTOR inhibitors have been used as medical therapy in lung carcinoids with variable results. No data are available on dopamine agonists as treatment for lung carcinoids. The main aim of the current study was to evaluate the effect of the combined treatment of somatostatin analogue octreotide and the dopamine agonist cabergoline with mTOR inhibitors in an in vitro model of typical lung carcinoids: the NCI-H727 cell line. In NCI-H727 cell line, reverse transcriptase-quantitative polymerase chain reaction and immunofluorescence were assessed to characterize the expression of the somatostatin receptor 2 and 5, dopamine receptor 2 and mTOR pathway components. Fifteen typical lung carcinoids tissue samples have been used for somatostatin receptor 2, dopamine receptor 2, and the main mTOR pathway component p70S6K expression and localization by immunohistochemistry. Cell viability, fluorescence-activated cell sorting analysis and western blot have been assessed to test the pharmacological effects of octreotide, cabergoline and mTOR inhibitors, and to evaluate the activation of specific cell signaling pathways in NCI-H727 cell line. NCI-H727 cell line expressed somatostatin receptor 2, somatostatin receptor 5 and dopamine receptor 2 and all mTOR pathway components at messenger and protein levels. Somatostatin receptor 2, dopamine receptor 2, and p70S6K (non phosphorylated and phosphorylated) proteins were expressed in most typical lung carcinoids tissue samples. Octreotide and cabergoline did not reduce cell viability as single agents but, when combined with mTOR inhibitors, they potentiate mTOR inhibitors effect after long-term exposure, reducing Akt and ERK phosphorylation, mTOR escape mechanisms, and increasing the expression DNA-damage-inducible transcript 4, an mTOR suppressor. In conclusion, the single use of octreotide and cabergoline is not sufficient to block cell viability but the combined approach of these agents with mTOR inhibitors might reduce the mTOR inhibitors-induced escape mechanisms and/or activate the endogenous mTOR suppressor, potentiating the effect of the mTOR inhibitors in an in vitro model of typical lung carcinoids.

Interleukin-8 promotes canine hemangiosarcoma growth by regulating the tumor microenvironment

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

Kim, Jong-Hyuk, E-mail: jhkim@umn.edu; Masonic Cancer Center, University of Minnesota, Minneapolis, MN; Frantz, Aric M.

Interleukin-8 (IL-8) gene expression is highly up-regulated in canine hemangiosarcoma (HSA); however, its role in the pathogenesis of this disease is unknown. We investigated the expression of IL-8 in canine HSA tissues and cell lines, as well and the effects of IL-8 on canine HSA in vitro, and in vivo using a mouse xenograft model for the latter. Constitutive expression of IL-8 mRNA, IL-8 protein, and IL-8 receptor were variable among different tumor samples and cell lines, but they showed stable steady states in each cell line. Upon the addition of IL-8, HSA cells showed transient intracellular calcium fluxes, suggestingmore » that their IL-8 receptors are functional and that IL-8 binding activates relevant signaling pathways. Yet, neither addition of exogenous IL-8 nor blockade of endogenous IL-8 by neutralizing anti-IL-8 antibody (α-IL-8 Ab) affected HSA cell proliferation or survival in vitro. To assess potential effects of IL-8 in other tumor constituents, we stratified HSA cell lines and whole tumor samples into “IL-8 high” and “IL-8 low” groups. Genome-wide gene expression profiling showed that samples in the “IL-8 high” tumor group were enriched for genes associated with a “reactive microenvironment,” including activation of coagulation, inflammation, and fibrosis networks. Based on these findings, we hypothesized that the effects of IL-8 on these tumors were mostly indirect, regulating interactions with the microenvironment. This hypothesis was supported by in vivo xenograft experiments where survival and engraftment of tumor cells was inhibited by administration of neutralizing α-IL-8 Ab. Together, our results suggest that IL-8 contributes to establishing a permissive microenvironment during the early stages of tumorigenesis in HSA. - Highlights: • IL-8 is expressed in canine hemangiosarcoma tumor samples and cell lines. • IL-8 transduces a relevant biological signal in canine hemangiosarcoma cells. • IL-8 gene signature is associated with reactive tumor microenvironments. • IL-8 potentiates tumor cell survival and engraftment into host tissues. • Canine hemangiosarcoma provides a unique comparative model for IL-8 studies.« less

Endogenous channels in HEK cells and potential roles in HCN ionic current measurements.

PubMed

Varghese, Anthony; Tenbroek, Erica M; Coles, James; Sigg, Daniel C

2006-01-01

A transformed line of human embryonic kidney epithelial cells (HEK 293) is commonly used as an expression system for exogenous ion channel genes. Previously, it has been shown that these cells contain mRNAs for a variety of ion channels. Expression of some of these genes has been confirmed at the protein level. Patch-clamp electrophysiology experiments confirm the presence of multiple ion channels and molecular data agree with pharmacological profiles of identified channels. In this work, we show that endogenous voltage-gated potassium channels in HEK cells are a significant source of outward current at positive potentials. We show that both non-transfected HEK cells and HEK cells transfected with hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have a significant amount of voltage-gated potassium (K(V)) current when certain tail current voltage-clamp protocols are used to assay HCN current activation. Specifically, tail current protocols that use a depolarized holding potential of -40 mV followed by hyperpolarizing pulses (-80 to -140 mV) and then a tail pulse potential of +20 mV indicate K(V) channels undergo closed-state inactivation at the more depolarized holding potential of -40 mV, followed by recovery from inactivation (but no activation) at hyperpolarizing potentials and high amount of activation at the positive tail potential. Our results indicate that pulse protocols with positive tail pulses are inaccurate assays for HCN current in certain HEK cells. Surprisingly, HEK-293 cells were found to contain mRNA for HCN2 and HCN3 although we have not detected a significant and consistent endogenous I(f)-like current in these cells.

Competing endogenous RNA network crosstalk reveals novel molecular markers in colorectal cancer.

PubMed

Samir, Nehal; Matboli, Marwa; El-Tayeb, Hanaa; El-Tawdi, Ahmed; Hassan, Mohmed K; Waly, Amr; El-Akkad, Hesham A E; Ramadan, Mohamed G; Al-Belkini, Tarek N; El-Khamisy, Sherif; El-Asmar, Farid

2018-05-08

The competing endogenous RNA networks play a pivotal role in cancer diagnosis and progression. Novel properstrategies for early detection of colorectal cancer (CRC) are strongly needed. We investigated a novel CRC-specific RNA-based integrated competing endogenous network composed of lethal3 malignant brain tumor like1 (L3MBTL1) gene, long non-coding intergenic RNA- (lncRNA RP11-909B2.1) and homo sapiens microRNA-595 (hsa-miRNA-595) using in silico data analysis. RT-qPCR-based validation of the network was achieved in serum of 70 patients with CRC, 40 patients with benign colorectal neoplasm, and 20 healthy controls. Moreover, in cancer tissues of 20 of the 70 CRC cases were involved in the study. The expression of RNA-based biomarker network in both CRC and adjacent non-tumor tissues and their correlation with the serum levels of this network members was investigated. Lastly, the expression levels of the chosen ceRNA was verified in CRC cell line. Our results revealed that the three RNAs-based biomarker network (long non-coding intergenic RNA-[lncRNA RP11-909B2.1], Homo sapiens microRNA-595 [hsa-miRNA-595], and L3MBTL1 mRNA), had high sensitivity and specificity for discriminating CRC from healthy controls and also from benign colorectal neoplasm. The data suggest that among these three RNAs, serum lncRNA RP11-909B2.1 could be a promising independent prognostic factors in CRC. The circulatory RNA based biomarker panel can act as potential biomarker for CRC diagnosis and prognosis. © 2018 Wiley Periodicals, Inc.

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells.

PubMed

Peters, Haley L; Tripathi, Satyendra C; Kerros, Celine; Katayama, Hiroyuki; Garber, Haven R; St John, Lisa S; Federico, Lorenzo; Meraz, Ismail M; Roth, Jack A; Sepesi, Boris; Majidi, Mourad; Ruisaard, Kathryn; Clise-Dwyer, Karen; Roszik, Jason; Gibbons, Don L; Heymach, John V; Swisher, Stephen G; Bernatchez, Chantale; Alatrash, Gheath; Hanash, Samir; Molldrem, Jeffrey J

2017-04-01

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases-induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319-29. ©2017 AACR . ©2017 American Association for Cancer Research.

The Netrin-4/ Neogenin-1 axis promotes neuroblastoma cell survival and migration

PubMed Central

Villanueva, Andrea A.; Falcón, Paulina; Espinoza, Natalie; Luis, Solano R.; Milla, Luis A.; Hernandez-SanMiguel, Esther; Torres, Vicente A.; Sanchez-Gomez, Pilar; Palma, Verónica

2017-01-01

Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling. PMID:28038459

Extracellular matrix and hormones transcriptionally regulate bovine. beta. -casein 5 prime sequences in stably transfected mouse mammary cells

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

Schmidhauser, C. Bissell, M.J.; Myers, C.A.; Casperson, G.F.

1990-12-01

Milk protein regulation involves synergistic action of lactogenic hormones and extracellular matrix (ECM). It is well established that substratum has a dramatic effect on morphology and function of mammary cells. The molecular mechanisms that regulate the ECM- and hormone-dependent gene expression, however, have not been resolved. To address this question, a subpopulation (designated CID 9) of the mouse mammary epithelial cell strain COMMA-2D has been developed in which more than 35% of the cells express {beta}-casein, form alveoli-like structures when plated onto a reconstituted basement membrane, and secrete {beta}-casein undirectionally into a lumen. These cells were stably transfected with amore » series of chloramphenicol acetyltransferase (CAT) fusion genes to study transcriptional regulation of the bovine {beta}-casein gene. The expression of CAT in these lines demonstrated a striking matrix and hormone dependency. This regulation occurered primarily at the transcriptional level and was dependent on the length of the 5{prime} flanking region of the {beta}-casein promotor. Both matrix and hormonal control of transcription occurred within at least the first 1790 base pairs upstream and/or 42 base pairs downstream of the transcriptional initiation site. The ECM effect was independent of glucocorticoid stimulation. However, prolactin was essential and hydrocortisone further increased CAT expression. Endogenous {beta}-casein expression in these lines was similar to that of the parent CID 9 cells. Our data indicate the existence of matrix-dependent elements that regulate transcription.« less

Investigation of TXNIP (thioredoxin-interacting protein) and TRX (thioredoxin) genes for growth-related traits in pigs.

PubMed

Yu, Mei; Geiger, Becky; Deeb, Nader; Rothschild, Max F

2007-03-01

It is well known that TRX and its endogenous inhibitor TXNIP help sustain the cellular reduction/oxidation balance in response to various stresses and both play a crucial role in cell proliferation and growth. Five SNPs were found in TXNIP and these allowed us to map it by linkage to SSC4. Three of the SNPs were used for association analyses in three commercial pig populations (Duroc, Hampshire, and synthetic line) with more than 1200 animals. Both the single-marker and haplotype analyses revealed significant effects of TXNIP on hot carcass weight, test daily gain, and lifetime daily gain. TRX was mapped on SSC1 but no significant associations with growth-related traits were found in the synthetic pig line in which the SNP was informative. The expression levels of TXNIP and TRX were then detected in two groups (fast growth and slow growth, respectively) with different genetic backgrounds for growth. Compared with the slow-growth group, TXNIP expression was significantly lower in the fast-growth group, whereas a marked increase in TRX expression was observed in fast-growth group. Our findings suggest that TXNIP has effects on growth-related traits in pigs and further investigations will be necessary to elucidate the underlying mechanisms involved.

C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein β-Glucuronidase on Dendritic Cells

PubMed Central

Mori, Daiki; Shibata, Kensuke; Yamasaki, Sho

2017-01-01

C-type lectin receptors (CLRs) recognize pathogen-derived ligands and abnormal self that trigger protective immune responses. However, the precise nature of self ligands recognized by CLRs remains to be determined. Here, we found that Dectin-2 recognizes bone marrow-derived dendritic cells (BMDCs) using Dectin-2-expressing reporter cells. This activity was inhibited by an excessive amount of mannose, and by the mutation of mannose-binding motif in Dectin-2. β-glucuronidase (Gusb) was identified as a protein bound to Dectin-2 and mutations of N-glycosylation sites in Gusb impaired the binding of Gusb to Dectin-2. Overexpression of Gusb in a macrophage cell line conferred an ability to stimulate Dectin-2-expressing reporter cells. Our study suggests that a glycosylated protein with mannose-related structure is recognized by Dectin-2. PMID:28046067

C-Type Lectin Receptor Dectin-2 Binds to an Endogenous Protein β-Glucuronidase on Dendritic Cells.

PubMed

Mori, Daiki; Shibata, Kensuke; Yamasaki, Sho

2017-01-01

C-type lectin receptors (CLRs) recognize pathogen-derived ligands and abnormal self that trigger protective immune responses. However, the precise nature of self ligands recognized by CLRs remains to be determined. Here, we found that Dectin-2 recognizes bone marrow-derived dendritic cells (BMDCs) using Dectin-2-expressing reporter cells. This activity was inhibited by an excessive amount of mannose, and by the mutation of mannose-binding motif in Dectin-2. β-glucuronidase (Gusb) was identified as a protein bound to Dectin-2 and mutations of N-glycosylation sites in Gusb impaired the binding of Gusb to Dectin-2. Overexpression of Gusb in a macrophage cell line conferred an ability to stimulate Dectin-2-expressing reporter cells. Our study suggests that a glycosylated protein with mannose-related structure is recognized by Dectin-2.

Endogenous Agmatine Induced by Ischemic Preconditioning Regulates Ischemic Tolerance Following Cerebral Ischemia

PubMed Central

Kim, Jae Hwan; Kim, Jae Young; Jung, Jin Young; Lee, Yong Woo; Lee, Won Taek; Huh, Seung Kon

2017-01-01

Ischemic preconditioning (IP) is one of the most important endogenous mechanisms that protect the cells against ischemia-reperfusion (I/R) injury. However, the exact molecular mechanisms remain unclear. In this study, we showed that changes in the level of agmatine were correlated with ischemic tolerance. Changes in brain edema, infarct volume, level of agmatine, and expression of arginine decarboxylase (ADC) and nitric oxide synthases (NOS; inducible NOS [iNOS] and neural NOS [nNOS]) were analyzed during I/R injury with or without IP in the rat brain. After cerebral ischemia, brain edema and infarct volume were significantly reduced in the IP group. The level of agmatine was increased before and during ischemic injury and remained elevated in the early reperfusion phase in the IP group compared to the experimental control (EC) group. During IP, the level of plasma agmatine was increased in the early phase of IP, but that of liver agmatine was abruptly decreased. However, the level of agmatine was definitely increased in the ipsilateral and contralateral hemisphere of brain during the IP. IP also increased the expression of ADC—the enzyme responsible for the synthesis of endogenous agmatine—before, during, and after ischemic injury. In addition, ischemic injury increased endogenous ADC expression in the EC group. The expression of nNOS was reduced in the I/R injured brain in the IP group. These results suggest that endogenous increased agmatine may be a component of the ischemic tolerance response that is induced by IP. Agmatine may have a pivotal role in endogenous ischemic tolerance. PMID:29302205

CREB-1 and AP-1 transcription factors JunD and Fra-2 regulate bone sialoprotein gene expression in human breast cancer cells.

PubMed

Detry, C; Lamour, V; Castronovo, V; Bellahcène, A

2008-02-01

Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a -56/-84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells.

Ectopic transgene expression in the retina of four transgenic mouse lines

PubMed Central

Gábriel, Robert; Erdélyi, Ferenc; Szabó, Gábor; Lawrence, J. Josh

2017-01-01

Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/ Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/ CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR-and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/ CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression. PMID:26563404

Decreased expression of RNA interference machinery, Dicer and Drosha, is associated with poor outcome in ovarian cancer patients

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

Merritt, William M.; Lin, Yvonne G.; Han, Liz Y.

2008-05-06

The clinical and functional significance of RNA interference (RNAi) machinery, Dicer and Drosha, in ovarian cancer is not known and was examined. Dicer and Drosha expression was measured in ovarian cancer cell lines (n=8) and invasive epithelial ovarian cancer specimens (n=111) and correlated with clinical outcome. Validation was performed with previously published cohorts of ovarian, breast, and lung cancer patients. Anti-Galectin-3 siRNA and shRNA transfections were used for in vitro functional studies. Dicer and Drosha mRNA and protein levels were decreased in 37% to 63% of ovarian cancer cell lines and in 60% and 51% of human ovarian cancer specimens,more » respectively. Low Dicer was significantly associated with advanced tumor stage (p=0.007), and low Drosha with suboptimal surgical cytoreduction (p=0.02). Tumors with both high Dicer and Drosha were associated with increased median patient survival (>11 years vs. 2.66 years for other groups; p<0.001). In multivariate analysis, high Dicer (HR=0.48; p=0.02), high-grade histology (HR=2.46; p=0.03), and poor chemoresponse (HR=3.95; p<0.001) were identified as independent predictors of disease-specific survival. Findings of poor clinical outcome with low Dicer expression were validated in separate cohorts of cancer patients. Galectin-3 silencing with siRNA transfection was superior to shRNA in cell lines with low Dicer (78-95% vs. 4-8% compared to non-targeting sequences), and similar in cell lines with high Dicer. Our findings demonstrate the clinical and functional impact of RNAi machinery alterations in ovarian carcinoma and support the use of siRNA constructs that do not require endogenous Dicer and Drosha for therapeutic applications.« less

Efficacy of ribavirin against malignant glioma cell lines: Follow-up study

PubMed Central

Ochiai, Yushi; Sano, Emiko; Okamoto, Yutaka; Yoshimura, Sodai; Makita, Kotaro; Yamamuro, Shun; Ohta, Takashi; Ogino, Akiyoshi; Tadakuma, Hisashi; Ueda, Takuya; Nakayama, Tomohiro; Hara, Hiroyuki; Yoshino, Atsuo; Katayama, Yoichi

2018-01-01

Ribavirin, a nucleic acid analog, has been employed as an antiviral agent against RNA and DNA viruses and has become the standard agent used for chronic hepatitis C in combination with interferon-α2a. Furthermore, the potential antitumor efficacy of ribavirin has attracted increasing interest. Recently, we demonstrated a dose-dependent antitumor effect of ribavirin for seven types of malignant glioma cell lines. However, the mechanism underlying the antitumor effect of ribavirin has not yet been fully elucidated. Therefore, the main aim of the present study was to provide further relevant data using two types of malignant glioma cell lines (U-87MG and U-138MG) with different expression of MGMT. Dotted accumulations of γH2AX were found in the nuclei and increased levels of ATM and phosphorylated ATM protein expression were also observed following ribavirin treatment (10 µM of ribavirin, clinical relevant concentration) in both the malignant glioma cells, indicating double-strand breaks as one possible mechanism underlying the antitumor effect of ribavirin. In addition, based on assessements using FACS, ribavirin treatment tended to increase the G0/G1 phase, with a time-lapse, indicating the induction of G0/G1-phase arrest. Furthermore, an increased phosphorylated p53 and p21 protein expression was confirmed in both glioma cells. Additionally, analysis by FACS indicated that apoptosis was induced following ribavirin treatment and caspase cascade, downstream of the p53 pathway, which indicated the activation of both exogenous and endogenous apoptosis in both malignant glioma cell lines. These findings may provide an experimental basis for the clinical treatment of glioblastomas with ribavirin. PMID:29251333

S100A8/A9 regulates MMP-2 expression and invasion and migration by carcinoma cells

PubMed Central

Silva, Emmanuel J.; Argyris, Prokopios P.; Zou, Xianqiong; Ross, Karen F.; Herzberg, Mark C.

2014-01-01

Intracellular calprotectin (S100A8/A9) functions in the control of the cell cycle checkpoint at G2/M. Dysregulation of S100A8/A9 appears to cause loss of the checkpoint, which frequently characterizes head and neck squamous cell carcinoma (HNSCC). In the present study, we analyzed carcinoma cells for other S100A8/A9-directed changes in malignant phenotype. Using a S100A8/A9-negative human carcinoma cell line (KB), transfection to express S100A8 and S100A9 caused selective down-regulation of MMP-2 and inhibited in vitro invasion and migration. Conversely, silencing of endogenous S100A8 and S100A9 expression in TR146 cells, a well-differentiated HNSCC cell line, increased MMP-2 activity and in vitro invasion and migration. When MMP-2 expression was silenced, cells appeared to assume a less malignant phenotype. To more closely model the architecture of cell growth in vivo, cells were grown in a 3D collagen substrate, which was compared to 2D. Growth on 3D substrates caused greater MMP-2 expression. Whereas hypermethylation of CpG islands occurs frequently in HNSCC, S100A8/A9-dependent regulation of MMP-2 could not be explained by modification of the upstream promoters of MMP2 or TIMP2. Collectively, these results suggest that intracellular S100A8/A9 contributes to the cancer cell phenotype by modulating MMP-2 expression and activity to regulate cell migration and mobility. PMID:25236491

Cells redox environment modulates BRCA1 expression and DNA homologous recombination repair.

PubMed

Wilson, Aaron; Yakovlev, Vasily A

2016-12-01

Cancer development and progression have been linked to oxidative stress, a condition characterized by unbalanced increase in ROS and RNS production. The main endogenous initiators of the redox imbalance in cancer cells are defective mitochondria, elevated NOX activity, and uncoupled NOS3. Traditionally, most attention has been paid to direct oxidative damage to DNA by certain ROS. However, increase in oxidative DNA lesions does not always lead to malignancy. Hence, additional ROS-dependent, pro-carcinogenic mechanisms must be important. Our recent study demonstrated that Tyr nitration of PP2A stimulates its activity and leads to downregulation of BRCA1 expression. This provides a mechanism for chromosomal instability essential for tumor progression. In the present work, we demonstrated that inhibition of ROS production by generating mitochondrial-electron-transport-deficient cell lines (ρ 0 cells) or by inhibition of NOX activity with a selective peptide inhibitor significantly reduced PP2A Tyr nitration and its activity in different cancer cell lines. As a result of the decreased PP2A activity, BRCA1 expression was restored along with a significantly enhanced level of DNA HRR. We used TCGA database to analyze the correlation between expressions of the NOX regulatory subunits, NOS isoforms, and BRCA1 in the 3 cancer research studies: breast invasive carcinoma, ovarian cystadenocarcinoma, and lung adenocarcinoma. TCGA database analysis demonstrated that the high expression levels of most of the NOX regulatory subunits responsible for stimulation of NOX1-NOX4 were associated with significant downregulation of BRCA1 expression. Copyright © 2016. Published by Elsevier Inc.

Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

PubMed

Dzieran, Johanna; Fabian, Jasmin; Feng, Teng; Coulouarn, Cédric; Ilkavets, Iryna; Kyselova, Anastasia; Breuhahn, Kai; Dooley, Steven; Meindl-Beinker, Nadja M

2013-01-01

Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

Apolipoprotein E promotes lipid accumulation and differentiation in human adipocytes

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

Lasrich, Dorothee; Bartelt, Alexander; Grewal, Thomas, E-mail: thomas.grewal@sydney.edu.au

Several studies in mice indicate a role for apolipoprotein E (APOE) in lipid accumulation and adipogenic differentiation in adipose tissue. However, little is yet known if APOE functions in a similar manner in human adipocytes. This prompted us to compare lipid loading and expression of adipocyte differentiation markers in APOE-deficient and control adipocytes using the differentiated human mesenchymal stem cell line hMSC-Tert as well as primary human and mouse adipocytes as model systems. Differentiated hMSC-Tert were stably transduced with or without siRNA targeting APOE while murine adipocytes were isolated from wild type and Apoe knockout mice. Human APOE knockdown hMSC-Tertmore » adipocytes accumulated markedly less triglycerides compared to control cells. This correlated with strongly decreased gene expression levels of adipocyte markers such as adiponectin (ADIPOQ) and fatty acid binding protein 4 (FABP4) as well as the key transcription factor driving adipocyte differentiation, peroxisome proliferator activator receptor gamma (PPARG), in particular the PPARG2 isoform. Similarly, differentiation of murine Apoe-deficient adipocytes was characterized by reduced gene expression of Adipoq, Fabp4 and Pparg. Interestingly, incubation of APOE-deficient hMSC-Tert adipocytes with conditioned media from APOE3-overexpressing adipocytes or APOE-containing Very Low Density Lipoprotein (VLDL) partially restored triglyceride accumulation, but were unable to induce adipocyte differentiation, as judged by expression of adipocyte markers. Taken together, depletion of endogenous APOE in human adipocytes severely impairs lipid accumulation, which is associated with an inability to initiate differentiation. - Highlights: • Immortalized human mesenchymal stem cells were used to study adipocyte development. • Knockdown of endogenous APOE lead to impaired lipid accumulation and adipogenesis. • APOE supplementation partially restored lipid accumulation but not differentiation. • Findings suggest dual functions of APOE for lipid accumulation and differentiation.« less

PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization

PubMed Central

Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

2016-01-01

PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways. PMID:26716897

Truncated EphA2 likely potentiates cell adhesion via integrins as well as infiltration and/or lodgment of a monocyte/macrophage cell line in the red pulp and marginal zone of the mouse spleen, where ephrin-A1 is prominently expressed in the vasculature.

PubMed

Konda, Naoko; Saeki, Noritaka; Nishino, Shingo; Ogawa, Kazushige

2017-03-01

We previously established a J774.1 monocyte/macrophage subline expressing a truncated EphA2 construct lacking the kinase domain. We demonstrated that following ephrin-A1 stimulation, endogenous EphA2 promotes cell adhesion through interaction with integrins and integrin ligands such as ICAM1 and that truncated EphA2 potentiates the adhesion and becomes associated with the integrin/integrin ligand complex. Based on these findings, we hypothesized that the EphA/ephrin-A system, particularly EphA2/ephrin-A1, regulates transendothelial migration/tissue infiltration of monocytes/macrophages, because ephrin-A1 is widely recognized to be upregulated in inflammatory vasculatures. To evaluate whether this hypothesis is applicable in the spleen, we screened for EphA2/ephrin-A1 expression and reexamined the cellular properties of the J774.1 subline. We found that ephrin-A1 was expressed in the vasculature of the marginal zone and the red pulp and that its expression was upregulated in response to phagocyte depletion; further, CD115, F4/80, and CXCR4 were expressed in J774.1 cells, which serve as a usable substitute for monocytes/macrophages. Moreover, following ephrin-A1 stimulation, truncated EphA2 did not detectably interfere with the phosphorylation of endogenous EphA2, and it potentiated cell adhesion possibly through modulation of integrin avidity. Accordingly, by intravenously injecting mice with equal numbers of J774.1 and the subline cells labeled with distinct fluorochromes, we determined that truncated EphA2 markedly potentiated preferential cell infiltration into the red pulp and the marginal zone. Thus, modulation of EphA2 signaling might contribute to effective transplantation of tissue-specific resident macrophages and/or monocytes.

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

PubMed Central

1995-01-01

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin. PMID:7642714

Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells.

PubMed

Yan, Z; DeGregori, J; Shohet, R; Leone, G; Stillman, B; Nevins, J R; Williams, R S

1998-03-31

Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized. We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene. Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line. We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.

Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

NASA Astrophysics Data System (ADS)

Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

2014-01-01

We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats.

PubMed

Melo, C H; Sousa, F C; Batista, R I P T; Sanchez, D J D; Souza-Fabjan, J M G; Freitas, V J F; Melo, L M; Teixeira, D I A

2015-07-31

The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to real-time PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia.

CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification.

PubMed

Friedel, Thorsten; Jung-Klawitter, Sabine; Sebe, Attila; Schenk, Franziska; Modlich, Ute; Ivics, Zoltán; Schumann, Gerald G; Buchholz, Christian J; Schneider, Irene C

2016-05-01

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.

Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

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

Silva, Patricio; Soto, Nicolás; Díaz, Jorge

2015-08-21

The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5more » is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.« less

Knockdown of Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 inhibits cell division and increases apoptosis in gastric cancer.

PubMed

Tang, Ze; He, Gan; Xu, Jie; Zhongfu, Li

2017-05-01

Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a pleiotropic protein associated with numerous cell functions, including transcription and differentiation. The role of CITED2 has been investigated in a number of malignancies; however, the roles of this protein in gastric cancers remain unclear. Therefore, we determined the role of CITED2 in gastric cancers. Gastric cancer cell lines (MKN74, MKN28, 7901, and AGS) were used to assess CITED2 transcript levels. Messenger RNA levels were determined using quantitative polymerase chain reaction. Lentiviral vectors containing CITED2 small interfering RNA were used to knockdown CITED2 expression. Cell proliferation was assessed with fluorescent imaging and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assays. Apoptosis and cell cycle stages were assessed through flow cytometry, and formation of colonies was determined using a fluorescent microscope. All cell lines tested in this study expressed CITED2. The cell line expressing the highest levels of CITED2 (MKN74) showed significant knockdown of endogenous CITED2 expression on lentiviral infection. Cell proliferation was shown to be lower in CITED2 knockdown MKN74 cells. G1/S-phase cell cycle arrest was observed on silencing of CITED2 in MKN74 cells. A significant increase in apoptosis was observed on CITED2 knock down in MKN74 cells, while colony forming ability was significantly inhibited after knock down of CITED2. CITED2 supports gastric cancer cell colony formation and proliferation while inhibiting apoptosis making it a potential gene therapy target for gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Markedly Increased Susceptibility to Natural Sheep Scrapie of Transgenic Mice Expressing Ovine PrP

PubMed Central

Vilotte, Jean-Luc; Soulier, Solange; Essalmani, Rachid; Stinnakre, Marie-George; Vaiman, Daniel; Lepourry, Laurence; Da Silva, Jose Costa; Besnard, Nathalie; Dawson, Mike; Buschmann, Anne; Groschup, Martin; Petit, Stephanie; Madelaine, Marie-Francoise; Rakatobe, Sabine; Le Dur, Annick; Vilette, Didier; Laude, Hubert

2001-01-01

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrPVRQ-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrPVRQ provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie. PMID:11390599

Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing.

PubMed

Roy, Ankita; Goodman, Joshua H; Begum, Gulnaz; Donnelly, Bridget F; Pittman, Gabrielle; Weinman, Edward J; Sun, Dandan; Subramanya, Arohan R

2015-02-15

Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.

MicroRNA-26b Enhances the Radiosensitivity of Hepatocellular Carcinoma Cells by Targeting EphA2.

PubMed

Jin, Qiao; Li, Xiang Jun; Cao, Pei Guo

2016-02-01

Sensitizing hepatocellular carcinoma (HCC) cells to irradiation is important to achieve satisfactory therapeutic effect with low-dose radiotherapy. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is a member of the Eph receptor family that constitutes the largest family of tyrosine kinase receptors. EphA2 overexpression is one of the poor prognostic factors in many progressive cancers. Importantly, EphA2 is a potential target of microRNA-26b (miR-26b), and miR-26b expression is down-regulated in several types of cancer. In this study, we measured the expression levels of miR-26b and EphA2 protein in seven human HCC cell lines by quantitative PCR and western blot analysis, respectively. Overall, lower miR-26b expression levels tended to be associated with higher EphA2 levels in HCC cell lines. Among the cell lines examined, 97H HCC cells expressed the lowest level of miR-26b and highest level of EphA2 protein. Thus, using 97H HCC cells, EphA2 mRNA was verified as the target of miR-26b by the luciferase reporter assay. Accordingly, a synthetic miR-26b, miR-26b mimics, was used to mimic the function of endogenous miR-26b. In 97H HCC cells transfected with miR-26b mimics or short-hairpin RNA targeting EphA2 mRNA, expression of EphA2 protein was reduced, which was associated with significantly lower proliferation rate and invasion ability and with higher apoptosis rate in response to low-dose irradiation, compared to control cells. In contrast, 97H HCC cells over-expressing EphA2 showed higher proliferation rate and invasion ability and lower apoptosis rate upon irradiation. These data suggest that miR-26b enhances the radiosensitivity of 97H HCC cells by targeting EphA2 protein.

Cysteine-Rich Intestinal Protein 1 Silencing Inhibits Migration and Invasion in Human Colorectal Cancer.

PubMed

He, Guoyang; Zou, Liyuan; Zhou, Lin; Gao, Peiqiong; Qian, Xinlai; Cui, Jing

2017-01-01

Cysteine-rich intestinal protein 1 (CRIP1), a member of the LIM/double zinc finger protein family, is abnormally expressed in several tumour types. However, few data are available on the role of CRIP1 in cancer. In the present study, we aimed to investigate the expression profile and functions of CRIP1 in colorectal cancer. To examine the protein expression level of CRIP1, immunohistochemistry (IHC) was performed on 56 pairs of colon cancer tissue samples. Western blotting was performed to investigate CRIP1 protein expression in four colon cancer cell lines. The endogenous expression of CRIP1 was suppressed using short interfering RNAs (siRNAs). Cell proliferation assays were used to determine whether CRIP1 silencing affected cell proliferation. Flow cytometry analysis was used to detect cell apoptosis. The effects of silencing CRIP1 on cell migration and invasion was detected using the transwell and wound-healing assays. IHC analysis showed that protein level of CRIP1 was significantly higher in tumour tissue samples than in paired non-tumour tissue samples and that the CRIP1 level was higher in metastatic tissue samples than in non-metastatic tissue samples. In addition, protein levels of CRIP1 were higher in highly metastatic colon cancer cell lines than in colon cancer cell lines with low metastasis. Further, CRIP1 silencing had no effect on cell proliferation or apoptosis in SW620 and HT29 cells. CRIP1 silencing suppressed cell migration and invasion obviously in SW620 and HT29 cells. The present study provides new evidence that abnormal expression of CRIP1 might be related to the degree of metastasis in colorectal cancer and that CRIP1 silencing could effectively inhibit migration and invasion during colorectal cancer development. These findings might aid the development of a biomarker for colon cancer prognosis and metastasis, and thus help to treat this common type of cancer. © 2017 The Author(s). Published by S. Karger AG, Basel.

Interferon-γ regulates chemokine expression and release in the human mast cell line HMC1: role of nitric oxide

PubMed Central

Gilchrist, M; Befus, A D

2008-01-01

Mast cells (MCs) are critical immune effector cells that release cytokines and chemokines involved in both homeostasis and disease. Interferon-γ (IFN-γ) is a pleiotropic cytokine that regulates multiple cellular activities. IFN-γ modulates rodent MC responsiveness via production of nitric oxide (NO), although the effects in human MC populations is unknown. We sought to investigate the effects of IFN-γ on expression of the chemokines interleukin-8 (IL-8) and CCL1 (I-309) in a human mast cell line (HMC1) and to determine the underlying regulatory mechanism. Nitric oxide synthase (NOS), IL-8 and CCL1 expression was determined using real-time polymerase chain reaction (PCR). NOS protein expression was analysed using western blot. NOS activity was determined using the citrulline assay. IL-8 and CCL1 release was measured by specific enzyme-linked immunosorbent assay (ELISA). IFN-γ inhibited phorbol 12-myristate 13-acetate (PMA)-induced release of IL-8 and CCL1 (by 47 and 38%). Real-time PCR analysis of IFN-γ-treated HMC1 showed a significant (P < 0·05) time-dependent increase in NOS1 and NOS3 mRNA. NOS3 protein was significantly increased at 18 hr, which correlated with a significant (P < 0·05) increase in constitutive NOS (cNOS) activity. IFN-γ-induced inhibition of chemokine expression and release was NO dependent, as treatment with the NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) reduced the IFN-γ inhibitory effect on IL-8 and CCL1 mRNA expression. NO donors mimicked the IFN-γ effect. IFN-γ inhibited PMA-induced cAMP response element binding protein (CREB) phosphorylation and DNA-binding activity. Our observations indicate for the first time that IFN-γ enhances endogenous NO formation through NOS3 activity, and that NO regulates the transcription and release of IL-8 and CCL1 in a human MC line. PMID:17662042

Validation of endogenous internal real-time PCR controls in renal tissues.

PubMed

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R; Mrug, Michal

2009-01-01

Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means. Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. Copyright 2009 S. Karger AG, Basel.

Uniconazole-induced starch accumulation in the bioenergy crop duckweed (Landoltia punctata) I: transcriptome analysis of the effects of uniconazole on chlorophyll and endogenous hormone biosynthesis.

PubMed

Liu, Yang; Fang, Yang; Huang, Mengjun; Jin, Yanling; Sun, Jiaolong; Tao, Xiang; Zhang, Guohua; He, Kaize; Zhao, Yun; Zhao, Hai

2015-01-01

Duckweed is a novel aquatic bioenergy crop that is found ubiquitously throughout the world. Uniconazole plays an important role in improving crop production through the regulation of endogenous hormone levels. We found that a high quantity and quality of duckweed growth can be achieved by uniconazole application, although the mechanisms are unknown. The fronds of Landoltia punctata were sprayed evenly with 800 mg/L uniconazole. The dry weight following treatment increased by 10% compared to the controls at 240 h. Endogenous cytokinin (CK) and abscisic acid (ABA) content both increased compared to the control, while the level of gibberellins (GAs) decreased. Additionally, gene expression profiling results showed that the expression of transcripts encoding key enzymes involved in endogenous CK and ABA biosynthesis were up-regulated, while the transcripts of key enzymes for GAs biosynthesis were down-regulated. On the other hand, chlorophyll a and chlorophyll b contents were both increased compared with the control. Moreover, the net photosynthetic rate was elevated to 25.6 μmol CO2/m(2)/s compared with the control value of 22.05 μmol CO2/m(2)/s. Importantly, the expression of some chlorophyll biosynthesis-related transcripts was up-regulated. Uniconazole treatment altered endogenous hormone levels and enhanced chlorophyll content and net photosynthetic rate in duckweed by regulating key enzymes involved in endogenous hormone and chlorophyll biosynthesis. The alterations of endogenous hormones and the increase of chlorophyll and photosynthetic rate data support the increase of biomass and starch accumulation.

Disease-specific induced pluripotent stem cells: a platform for human disease modeling and drug discovery.

PubMed

Jang, Jiho; Yoo, Jeong-Eun; Lee, Jeong-Ah; Lee, Dongjin R; Kim, Ji Young; Huh, Yong Jun; Kim, Dae-Sung; Park, Chul-Yong; Hwang, Dong-Youn; Kim, Han-Soo; Kang, Hoon-Chul; Kim, Dong-Wook

2012-03-31

The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.

C/EBPβ LIP and c-Jun synergize to regulate expression of the murine progesterone receptor.

PubMed

Wang, Weizhong; Do, Han Ngoc; Aupperlee, Mark D; Durairaj, Srinivasan; Flynn, Emily E; Miksicek, Richard J; Haslam, Sandra Z; Schwartz, Richard C

2018-06-02

CCAAT/enhancer binding protein β (C/EBPβ) is required for murine mammary ductal morphogenesis and alveologenesis. Progesterone is critical for proliferation and alveologenesis in adult mammary glands, and there is a similar requirement for progesterone receptor isoform B (PRB) in alveologenesis. We examined C/EBPβ regulation of PR expression. All three C/EBPβ isoforms, including typically inhibitory LIP, transactivated the PR promoter. LIP, particularly, strongly synergized with c-Jun to drive PR transcription. Endogenous C/EBPβ and c-Jun stimulated a PR promoter-reporter and these two factors showed promoter occupancy on the endogenous PR gene. Additionally, LIP overexpression elevated endogenous PR protein expression. In pregnancy, both PRB and the relative abundance of LIP among C/EBPβ isoforms increase. Consistent with a role in PRB expression, in vivo C/EBPβ and PR isoform A expression showed mutually exclusive localization in mammary epithelium, while C/EBPβ and PRB largely co-localized. We suggest a critical role for C/EBPβ, particularly LIP, in PRB expression. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

A role for NRAGE in NF-κB activation through the non-canonical BMP pathway

PubMed Central

2010-01-01

Background Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-κB has yet to be explored. Results Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -α/β and subsequent transcriptional activation of the p65 subunit of NF-κB. Ablation of endogenous NRAGE by siRNA inhibited NF-κB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-κB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor. Conclusion Modulation of NRAGE expression revealed novel roles in regulating NF-κB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway. PMID:20100315

The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed

PubMed Central

Filloux, Denis; Murrell, Sasha; Koohapitagtam, Maneerat; Golden, Michael; Julian, Charlotte; Galzi, Serge; Uzest, Marilyne; Rodier-Goud, Marguerite; D’Hont, Angélique; Vernerey, Marie Stephanie; Wilkin, Paul; Peterschmitt, Michel; Winter, Stephan; Murrell, Ben; Martin, Darren P.; Roumagnac, Philippe

2015-01-01

Endogenous viral sequences are essentially ‘fossil records’ that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21–24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species. PMID:27774276

Impaired thymic selection in mice expressing altered levels of the SLP-76 adaptor protein.

PubMed

Ramsey, Kimberley; Luckashenak, Nancy; Koretzky, Gary A; Clements, James L

2008-02-01

Intracellular signaling initiated by ligation of the TCR influences cell fate at multiple points during the lifespan of a T cell. This is especially evident during thymic selection, where the nature of TCR-dependent signaling helps to establish a MHC-restricted, self-tolerant T cell repertoire. The Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76) adaptor protein is a required intermediate in multiple signaling pathways triggered by TCR engagement, several of which have been implicated in dictating the outcome of thymic selection (e.g., intracellular calcium flux and activation of ERK family MAPKs). To determine if thymocyte maturation and selection at later stages of development are sensitive to perturbations in SLP-76 levels, we analyzed these crucial events using several transgenic (Tg) lines of mice expressing altered levels of SLP-76 in the thymus. In Tg mice expressing low levels of SLP-76 in preselection thymocytes, the CD4:CD8 ratio in the thymus and spleen was skewed in a manner consistent with impaired selection and/or maturation of CD4+ thymocytes. Low SLP-76 expression also correlated with reduced CD5 expression on immature thymocytes, consistent with reduced TCR signaling potential. In contrast, reconstitution of SLP-76 at higher levels resulted in normal thymic CD5 expression and CD4:CD8 ratios in the thymus and periphery. It is curious that thymic deletion of TCR-Tg (HY) thymocytes was markedly impaired in both lines of Tg-reconstituted SLP-76-/- mice. Studies using chimeric mice indicate that the defect in deletion of HY+ thymocytes is intrinsic to the developing thymocyte, suggesting that maintenance of sufficient SLP-76 expression from the endogenous locus is a key element in the selection process.

Downregulation of Bit1 expression promotes growth, anoikis resistance, and transformation of immortalized human bronchial epithelial cells via Erk activation-dependent suppression of E-cadherin.

PubMed

Yao, Xin; Gray, Selena; Pham, Tri; Delgardo, Mychael; Nguyen, An; Do, Stephen; Ireland, Shubha Kale; Chen, Renwei; Abdel-Mageed, Asim B; Biliran, Hector

2018-01-01

The mitochondrial Bit1 protein exerts tumor-suppressive function in NSCLC through induction of anoikis and inhibition of EMT. Having this dual tumor suppressive effect, its downregulation in the established human lung adenocarcinoma A549 cell line resulted in potentiation of tumorigenicity and metastasis in vivo. However, the exact role of Bit1 in regulating malignant growth and transformation of human lung epithelial cells, which are origin of most forms of human lung cancers, has not been examined. To this end, we have downregulated the endogenous Bit1 expression in the immortalized non-tumorigenic human bronchial epithelial BEAS-2B cells. Knockdown of Bit1 enhanced the growth and anoikis insensitivity of BEAS-2B cells. In line with their acquired anoikis resistance, the Bit1 knockdown BEAS-2B cells exhibited enhanced anchorage-independent growth in vitro but failed to form tumors in vivo. The loss of Bit1-induced transformed phenotypes was in part attributable to the repression of E-cadherin expression since forced exogenous E-cadherin expression attenuated the malignant phenotypes of the Bit1 knockdown cells. Importantly, we show that the loss of Bit1 expression in BEAS-2B cells resulted in increased Erk activation, which functions upstream to promote TLE1-mediated transcriptional repression of E-cadherin. These collective findings indicate that loss of Bit1 expression contributes to the acquisition of malignant phenotype of human lung epithelial cells via Erk activation-induced suppression of E-cadherin expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Reproductive performance assessment of two White Leghorn lines free of endogenous viruses

USDA-ARS?s Scientific Manuscript database

Two of the chicken lines that USDA-ARS Avian Disease & Oncology Laboratory (ADOL) have developed and maintained are line 0 and line 0.TVB*S1 (also known as rapid feathering susceptible or RFS). Line 0 was developed in the 1980s (Bacon et al., 2000), and RFS was developed in 2007 (Zhang et al., 2008)...

Recombinant probes for visualizing endogenous synaptic proteins in living neurons

PubMed Central

Gross, Garrett G.; Junge, Jason A.; Mora, Rudy J.; Kwon, Hyung-Bae; Olson, C. Anders; Takahashi, Terry T.; Liman, Emily R.; Ellis-Davies, Graham C.R.; McGee, Aaron W.; Sabatini, Bernardo L.; Roberts, Richard W.; Arnold, Don B.

2013-01-01

Summary The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed FingRs (Fibronectin intrabodies generated with mRNA display), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and which, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a novel transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo. PMID:23791193

LINE1 contributes to autoimmunity through both RIG-I- and MDA5-mediated RNA sensing pathways.

PubMed

Zhao, Ke; Du, Juan; Peng, Yanfeng; Li, Peng; Wang, Shaohua; Wang, Yu; Hou, Jingwei; Kang, Jian; Zheng, Wenwen; Hua, Shucheng; Yu, Xiao-Fang

2018-06-01

Improper host immune activation leads to the development of the autoimmune disease Aicardi-Goutières syndrome (AGS), which is attributed to defined genetic mutations in such proteins as TREX1 and ADAR1. The mechanism of immune activation in AGS patients has not been thoroughly elucidated to date. In this study, we report that endogenous LINE1 components trigger IFNβ production in multiple human cell types, including those defective for cGAS/STING-mediated DNA sensing. In these cells, LINE1 DNA synthesis and retrotransposition were not required for LINE1-triggered immune activation, but RNA sensing pathways were essential. LINE1-triggered immune activation could be suppressed by diverse LINE1 inhibitors, including AGS-associated proteins targeting LINE1 RNA or proteins. However, AGS-associated ADAR1 or TREX1 mutants were defective in suppressing LINE1 retrotransposition or LINE1-triggered immune activation. Therefore, we have revealed a new function for LINE1 as an endogenous trigger of innate immune activation, which is important for understanding the molecular basis of IFN-based autoimmune diseases and may offer new intervention strategies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pericytes of Multiple Organs Do Not Behave as Mesenchymal Stem Cells In Vivo.

PubMed

Guimarães-Camboa, Nuno; Cattaneo, Paola; Sun, Yunfu; Moore-Morris, Thomas; Gu, Yusu; Dalton, Nancy D; Rockenstein, Edward; Masliah, Eliezer; Peterson, Kirk L; Stallcup, William B; Chen, Ju; Evans, Sylvia M

2017-03-02

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of Secretory Odontoblasts Using DMP1-GFP Transgenic Mice

PubMed Central

Balic, Anamaria; Mina, Mina

2011-01-01

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue-and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts. PMID:21172466

OsSUV3 transgenic rice maintains higher endogenous levels of plant hormones that mitigates adverse effects of salinity and sustains crop productivity.

PubMed

Sahoo, Ranjan Kumar; Ansari, Mohammad Wahid; Tuteja, Renu; Tuteja, Narendra

2014-01-01

The SUV3 (suppressor of Var 3) gene encodes a DNA and RNA helicase, which is localized in the mitochondria. Plant SUV3 has not yet been characterized in detail. However, the Arabidopsis ortholog of SUV3 (AT4G14790) has been shown to be involved in embryo sac development. Previously, we have reported that rice SUV3 functions as DNA and RNA helicase and provides salinity stress tolerance by maintaining photosynthesis and antioxidant machinery. Here, we report further analysis of the transgenic OsSUV3 rice plants under salt stress. The transgenic OsSUV3 overexpressing rice T1 lines showed significantly higher endogenous content of plant hormones viz., gibberellic acid (GA3), zeatin (Z) and indole-3-acetic acid (IAA) in leaf, stem and root as compared to wild-type (WT), vector control (VC) and antisense (AS) plants under salt (200 mM NaCl) stress condition. A similar trend of endogenous plant hormones profile was also reflected in the T2 generation of OsSUV3 transgenic rice under defined parameters and stress condition. In response to stress, OsSUV3 rice plants maintained plant hormone levels that regulate the expression of several stress-induced genes and reduce adverse effects of salt on plant growth and development and therefore sustains crop productivity.

VlincRNAs controlled by retroviral elements are a hallmark of pluripotency and cancer.

PubMed

St Laurent, Georges; Shtokalo, Dmitry; Dong, Biao; Tackett, Michael R; Fan, Xiaoxuan; Lazorthes, Sandra; Nicolas, Estelle; Sang, Nianli; Triche, Timothy J; McCaffrey, Timothy A; Xiao, Weidong; Kapranov, Philipp

2013-07-22

The function of the non-coding portion of the human genome remains one of the most important questions of our time. Its vast complexity is exemplified by the recent identification of an unusual and notable component of the transcriptome - very long intergenic non-coding RNAs, termed vlincRNAs. Here we identify 2,147 vlincRNAs covering 10 percent of our genome. We show they are present not only in cancerous cells, but also in primary cells and normal human tissues, and are controlled by canonical promoters. Furthermore, vlincRNA promoters frequently originate from within endogenous retroviral sequences. Strikingly, the number of vlincRNAs expressed from endogenous retroviral promoters strongly correlates with pluripotency or the degree of malignant transformation. These results suggest a previously unknown connection between the pluripotent state and cancer via retroviral repeat-driven expression of vlincRNAs. Finally, we show that vlincRNAs can be syntenically conserved in humans and mouse and their depletion using RNAi can cause apoptosis in cancerous cells. These intriguing observations suggest that vlincRNAs could create a framework that combines many existing short ESTs and lincRNAs into a landscape of very long transcripts functioning in the regulation of gene expression in the nucleus. Certain types of vlincRNAs participate at specific stages of normal development and, based on analysis of a limited set of cancerous and primary cell lines, they appear to be co-opted by cancer-associated transcriptional programs. This provides additional understanding of transcriptome regulation during the malignant state, and could lead to additional targets and options for its reversal.

IGFBP2 is a candidate biomarker for Ink4a-Arf status and a therapeutic target for high-grade gliomas.

PubMed

Moore, Lynette M; Holmes, Kristen M; Smith, Sarah M; Wu, Ying; Tchougounova, Elena; Uhrbom, Lene; Sawaya, Raymond; Bruner, Janet M; Fuller, Gregory N; Zhang, Wei

2009-09-29

The levels of insulin-like growth factor-binding protein 2 (IGFBP2) are elevated during progression of many human cancers. By using a glial-specific transgenic mouse system (RCAS/Ntv-a), we reported previously that IGFBP2 is an oncogenic factor for glioma progression in combination with platelet-derived growth factor-beta (PDGFB). Because the INK4a-ARF locus is often deleted in high-grade gliomas (anaplastic oligodendroglioma and glioblastoma), we investigated the effect of the Ink4a-Arf-null background on IGFBP2-mediated progression of PDGFB-initiated oligodendroglioma. We demonstrate here that homozygous deletion of Ink4a-Arf bypasses the requirement of exogenously introduced IGFBP2 for glioma progression. Instead, absence of Ink4a-Arf resulted in elevated endogenous tumor cell IGFBP2. An inverse relationship between p16(INK4a) and IGFBP2 expression was also observed in human glioma tissue samples and in 90 different cancer cell lines by using Western blotting and reverse-phase protein lysate arrays. When endogenous IGFBP2 expression was attenuated by an RCAS vector expressing antisense IGFBP2 in our mouse model, a decreased incidence of anaplastic oligodendroglioma as well as prolonged survival was observed. Thus, p16(INK4a) is a negative regulator of the IGFBP2 oncogene. Loss of Ink4a-Arf results in increased IGFBP2, which contributes to glioma progression, thereby implicating IGFBP2 as a marker and potential therapeutic target for Ink4a-Arf-deleted gliomas.

IGFBP2 is a candidate biomarker for Ink4a-Arf status and a therapeutic target for high-grade gliomas

PubMed Central

Moore, Lynette M.; Holmes, Kristen M.; Smith, Sarah M.; Wu, Ying; Tchougounova, Elena; Uhrbom, Lene; Sawaya, Raymond; Bruner, Janet M.; Fuller, Gregory N.; Zhang, Wei

2009-01-01

The levels of insulin-like growth factor-binding protein 2 (IGFBP2) are elevated during progression of many human cancers. By using a glial-specific transgenic mouse system (RCAS/Ntv-a), we reported previously that IGFBP2 is an oncogenic factor for glioma progression in combination with platelet-derived growth factor-β (PDGFB). Because the INK4a-ARF locus is often deleted in high-grade gliomas (anaplastic oligodendroglioma and glioblastoma), we investigated the effect of the Ink4a-Arf-null background on IGFBP2-mediated progression of PDGFB-initiated oligodendroglioma. We demonstrate here that homozygous deletion of Ink4a-Arf bypasses the requirement of exogenously introduced IGFBP2 for glioma progression. Instead, absence of Ink4a-Arf resulted in elevated endogenous tumor cell IGFBP2. An inverse relationship between p16INK4a and IGFBP2 expression was also observed in human glioma tissue samples and in 90 different cancer cell lines by using Western blotting and reverse-phase protein lysate arrays. When endogenous IGFBP2 expression was attenuated by an RCAS vector expressing antisense IGFBP2 in our mouse model, a decreased incidence of anaplastic oligodendroglioma as well as prolonged survival was observed. Thus, p16INK4a is a negative regulator of the IGFBP2 oncogene. Loss of Ink4a-Arf results in increased IGFBP2, which contributes to glioma progression, thereby implicating IGFBP2 as a marker and potential therapeutic target for Ink4a-Arf-deleted gliomas. PMID:19805356

Fasting modulates GH/IGF-I axis and its regulatory systems in the mammary gland of female mice: Influence of endogenous cortistatin.

PubMed

Villa-Osaba, Alicia; Gahete, Manuel D; Cordoba-Chacon, José; de Lecea, Luis; Castaño, Justo P; Luque, Raúl M

2016-10-15

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are essential factors in mammary-gland (MG) development and are altered during fasting. However, no studies have investigated the alterations in the expression of GH/IGF-I and its regulatory systems (somatostatin/cortistatin and ghrelin) in MG during fasting. Therefore, this study was aimed at characterizing the regulation of GH/IGF-I/somatostatin/cortistatin/ghrelin-systems expression in MG of fasted female-mice (compared to fed-controls) and the influence of endogenous-cortistatin (using cortistatin-knockouts). Fasting decreased IGF-I while increased IGF-I/Insulin-receptors expression in MGs. Fasting provoked an increase in GH expression that might be associated to enhanced ghrelin-variants/ghrelin-O-acyl-transferase enzyme expression, while an upregulation of somatostatin-receptors was observed. However, cortistatin-knockouts mice showed a decrease in GH and somatostatin receptor-subtypes expression. Altogether, we demonstrate that GH/IGF-I, somatostatin/cortistatin and ghrelin systems expression is altered in MG during fasting, suggesting a relevant role in coordinating its response to metabolic stress, wherein endogenous cortistatin might be essential for an appropriate response. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

N-acetyltransferase 2 activity and folate levels

PubMed Central

Cao, Wen; Strnatka, Diana; McQueen, Charlene A.; Hunter, Robert J.; Erickson, Robert P.

2010-01-01

Aims To determine whether increased N-acetyltransferase (NAT) activity might have a toxic effect during development and an influence on folate levels since previous work has shown that only low levels of exogenous NAT can be achieved in constitutionally transgenic mice (Cao, et al, 2005) Main Methods A human NAT1 tet-inducible construct was used that would not be expressed until the inducer was delivered. Human NAT1 cDNA was cloned into pTRE2 and injected into mouse oocytes. Two transgenic lines were crossed to mouse line TgN(rtTahCMV)4Uh containing the CMV promoted “teton.”Measurements of red blood cell folate levels in inbred strains of mice were performed. Key findings Only low levels of human NAT1 could be achieved in kidney (highly responsive in other studies) whether the inducer, doxycycline, was given by gavage or in drinking water.An inverse correlation of folate levels with Nat2 enzyme activity was found. Significance Since increasing NAT1 activity decrease folate in at least one tissue, the detrimental effect of expression of human NAT1 in combination with endogenous mouse Nat2 may be a consequence of increased catabolism of folate. PMID:19932120

I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

PubMed

Fenina, Myriam; Simon-Chazottes, Dominique; Vandormael-Pournin, Sandrine; Soueid, Jihane; Langa, Francina; Cohen-Tannoudji, Michel; Bernard, Bruno A; Panthier, Jean-Jacques

2012-01-01

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress

PubMed Central

Alet, Analía I; Sanchez, Diego H; Cuevas, Juan C; del Valle, Secundino; Altabella, Teresa; Tiburcio, Antonio F; Marco, Francisco; Ferrando, Alejandro; Espasandín, Fabiana D; González, María E; Carrasco, Pedro

2011-01-01

Polyamines have been globally associated to plant responses to abiotic stress. Particularly, putrescine has been related to a better response to cold and dehydration stresses. It is known that this polyamine is involved in cold tolerance, since Arabidopsis thaliana plants mutated in the key enzyme responsible for putrescine synthesis (arginine decarboxilase, ADC; EC 4.1.1.19) are more sensitive than the wild type to this stress. Although it is speculated that the overexpression of ADC genes may confer tolerance, this is hampered by pleiotropic effects arising from the constitutive expression of enzymes from the polyamine metabolism. Here, we present our work using A. thaliana transgenic plants harboring the ADC gene from oat under the control of a stress-inducible promoter (pRD29A) instead of a constitutive promoter. The transgenic lines presented in this work were more resistant to both cold and dehydration stresses, associated with a concomitant increment in endogenous putrescine levels under stress. Furthermore, the increment in putrescine upon cold treatment correlates with the induction of known stress-responsive genes, and suggests that putrescine may be directly or indirectly involved in ABA metabolism and gene expression. PMID:21330789

Recognition and killing of brain tumor stem-like initiating cells by CD8+ cytolytic T cells.

PubMed

Brown, Christine E; Starr, Renate; Martinez, Catalina; Aguilar, Brenda; D'Apuzzo, Massimo; Todorov, Ivan; Shih, Chu-Chih; Badie, Behnam; Hudecek, Michael; Riddell, Stanley R; Jensen, Michael C

2009-12-01

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

PubMed Central

Trkola, Alexandra; Matthews, Jamie; Gordon, Cynthia; Ketas, Tom; Moore, John P.

1999-01-01

We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies. PMID:10516002

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

PubMed

Endlich, B; Salavati, R; Sullivan, T; Ling, C C

1992-12-01

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors

PubMed Central

Rycaj, Kiera; Plummer, Joshua B.; Li, Ming; Yin, Bingnan; Frerich, Katherine; Garza, Jeremy G.; Shen, Jianjun; Lin, Kevin; Yan, Peisha; Glynn, Sharon A.; Dorsey, Tiffany H.; Hunt, Kelly K.; Ambs, Stefan; Johanning, Gary L.

2012-01-01

Background The envelope (env) protein of the human endogenous retrovirus type K (HERV-K) family is commonly expressed on the surface of breast cancer cells. We assessed whether HERV-K env is a potential target for antibody-based immunotherapy of breast cancer. Methods We examined the expression of HERV-K env protein in various malignant (MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1) and nonmalignant (MCF-10A and MCF-10AT) human breast cell lines by immunoblot, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry. Anti-HERV-K env monoclonal antibodies (mAbs; 6H5, 4D1, 4E11, 6E11, and 4E6) were used to target expression of HERV-K, and antitumor effects were assessed by quantifying growth and apoptosis of breast cancer cells in vitro, and tumor growth in vivo in mice (n = 5 per group) bearing xenograft tumors. The mechanisms responsible for 6H5 mAb–mediated effects were investigated by microarray assays, flow cytometry, immunoblot, and immunofluorescence staining. The expression of HERV-K env protein was assessed in primary breast tumors (n = 223) by immunohistochemistry. All statistical tests were two-sided. Results The expression of HERV-K env protein in malignant breast cancer cell lines was substantially higher than nonmalignant breast cells. Anti–HERV-K-specific mAbs inhibited growth and induced apoptosis of breast cancer cells in vitro. Mice treated with 6H5 mAb showed statistically significantly reduced growth of xenograft tumors compared with mice treated with control immunoglobulin (control [mIgG] vs 6H5 mAb, for tumors originating from MDA-MB-231 cells, mean size = 1448.33 vs 475.44 mm3; difference = 972.89 mm3, 95% CI = 470.17 to 1475.61 mm3; P < .001). Several proteins involved in the apoptotic signaling pathways were overexpressed in vitro in 6H5 mAb–treated malignant breast cells compared with mIgG-treated control. HERV-K expression was detected in 148 (66%) of 223 primary breast tumors, and a higher rate of lymph node metastasis was associated with HERV-K-positive compared with HERV-K-negative tumors (43% vs 23%, P = .003). Conclusion Monoclonal antibodies against HERV-K env protein show potential as novel immunotherapeutic agents for breast cancer therapy. PMID:22247020

Pathogenic role for macrophage migration inhibitory factor in glioblastoma and its targeting with specific inhibitors as novel tailored therapeutic approach

PubMed Central

Mangano, Katia; Mazzon, Emanuela; Basile, Maria Sofia; Di Marco, Roberto; Bramanti, Placido; Mammana, Santa; Petralia, Maria Cristina; Fagone, Paolo; Nicoletti, Ferdinando

2018-01-01

Macrophage Migration Inhibitory Factor (MIF) is a pro-inflammatory cytokine expressed by a variety of cell types. Although MIF has been primarily studied for its role in the pathogenesis of autoimmune diseases, it has also been shown to promote tumorigenesis and it is over expressed in various malignant tumors. MIF is able to induce angiogenesis, cell cycle progression, and to block apoptosis. As tailored therapeutic approaches for the inhibition of endogenous MIF are being developed, it is important to evaluate the role of MIF in individual neoplastic conditions that may benefit from specific MIF inhibitors. Along with this line, in this paper, we have reviewed the evidence of the involvement of MIF in the etiopathogenesis and progression of glioblastoma and the preclinical data suggesting the possible use of specific MIF inhibition as a potential novel therapeutic strategy for brain tumors. PMID:29707160

Derivation and Characterization of Induced Pluripotent Stem Cells from Equine Fibroblasts

PubMed Central

Breton, Amandine; Sharma, Ruchi; Diaz, Andrea Catalina; Parham, Alea Gillian; Graham, Audrey; Neil, Claire; Whitelaw, Christopher Bruce; Milne, Elspeth

2013-01-01

Pluripotent stem cells offer unprecedented potential not only for human medicine but also for veterinary medicine, particularly in relation to the horse. Induced pluripotent stem cells (iPSCs) are particularly promising, as they are functionally similar to embryonic stem cells and can be generated in vitro in a patient-specific manner. In this study, we report the generation of equine iPSCs from skin fibroblasts obtained from a foal and reprogrammed using viral vectors coding for murine Oct4, Sox2, c-Myc, and Klf4 sequences. The reprogrammed cell lines were morphologically similar to iPSCs reported from other species and could be stably maintained over more than 30 passages. Immunostaining and polymerase chain reaction analyses revealed that these cell lines expressed an array of endogenous markers associated with pluripotency, including OCT4, SOX2, NANOG, REX1, LIN28, SSEA1, SSEA4, and TRA1-60. Furthermore, under the appropriate conditions, the equine iPSCs readily formed embryoid bodies and differentiated in vitro into cells expressing markers of ectoderm, mesoderm, and endoderm, and when injected into immunodeficient mice, gave raise to tumors containing differentiated derivatives of the 3 germ layers. Finally, we also reprogrammed fibroblasts from a 2-year-old horse. The reprogrammed cells were similar to iPSCs derived from neonatal fibroblasts in terms of morphology, expression of pluripotency markers, and differentiation ability. The generation of these novel cell lines constitutes an important step toward the understanding of pluripotency in the horse, and paves the way for iPSC technology to potentially become a powerful research and clinical tool in veterinary biomedicine. PMID:22897112

A Raf-competitive K-Ras binder can fail to functionally antagonize signaling.

PubMed

Kauke, Monique J; Tisdale, Alison W; Kelly, Ryan L; Braun, Christian J; Hemann, Michael T; Wittrup, K Dane

2018-05-02

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically-encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacological efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Copyright ©2018, American Association for Cancer Research.

Grouper tshβ Promoter-Driven Transgenic Zebrafish Marks Proximal Kidney Tubule Development

PubMed Central

Wang, Yang; Sun, Zhi-Hui; Zhou, Li; Li, Zhi; Gui, Jian-Fang

2014-01-01

Kidney tubule plays a critical role in recovering or secreting solutes, but the detailed morphogenesis remains unclear. Our previous studies have found that grouper tshβ (gtshβ) is also expressed in kidney, however, the distribution significance is still unknown. To understand the gtshβ role and kidney tubule morphogenesis, here, we have generated a transgenic zebrafish line Tg(gtshβ:GFP) with green fluorescent protein driven by the gtshβ promoter. Similar to the endogenous tshβ in zebrafish or in grouper, the gtshβ promoter-driven GFP is expressed in pituitary and kidney, and the developing details of proximal kidney tubule are marked in the transgenic zebrafish line. The gfp initially transcribes at 16 hours post fertilization (hpf) above the dorsal mesentery, and partially co-localizes with pronephric tubular markers slc20a1a and cdh17. Significantly, the GFP specifically localizes in proximal pronephric segments during embryogenesis and resides at kidney duct epithelium in adult fish. To test whether the gtshβ promoter-driven GFP may serve as a readout signal of the tubular development, we have treated the embryos with retinoic acid signaing (RA) reagents, in which exogenous RA addition results in a distal extension of the proximal segments, while RA inhibition induces a weakness and shortness of the proximal segments. Therefore, this transgenic line provides a useful tool for genetic or chemical analysis of kidney tubule. PMID:24905828

Feeder & basic fibroblast growth factor-free culture of human embryonic stem cells: Role of conditioned medium from immortalized human feeders.

PubMed

Teotia, Pooja; Sharma, Shilpa; Airan, Balram; Mohanty, Sujata

2016-12-01

Human embryonic stem cell (hESC) lines are commonly maintained on inactivated feeder cells, in the medium supplemented with basic fibroblast growth factor (bFGF). However, limited availability of feeder cells in culture, and the high cost of growth factors limit their use in scalable expansion of hESC cultures for clinical application. Here, we describe an efficient and cost-effective feeder and bFGF-free culture of hESCs using conditioned medium (CM) from immortalized feeder cells. KIND-1 hESC cell line was cultured in CM, collected from primary mouse embryonic fibroblast, human foreskin fibroblast (HFF) and immortalized HFF (I-HFF). Pluripotency of KIND-1 hESC cell line was confirmed by expression of genes, proteins and cell surface markers. In culture, these cells retained normal morphology, expressed all cell surface markers, could differentiate to embryoid bodies upon culture in vitro. Furthermore, I-HFF feeder cells without supplementation of bFGF released ample amount of endogenous bFGF to maintain stemness of hESC cells. The study results described the use of CM from immortalized feeder cells as a consistent source and an efficient, inexpensive feeder-free culture system for the maintenance of hESCs. Moreover, it was possible to maintain hESCs without exogenous supplementation of bFGF. Thus, the study could be extended to scalable expansion of hESC cultures for therapeutic purposes.

Heat shock protein 90-mediated peptide-selective presentation of cytosolic tumor antigen for direct recognition of tumors by CD4(+) T cells.

PubMed

Tsuji, Takemasa; Matsuzaki, Junko; Caballero, Otavia L; Jungbluth, Achim A; Ritter, Gerd; Odunsi, Kunle; Old, Lloyd J; Gnjatic, Sacha

2012-04-15

Tumor Ag-specific CD4(+) T cells play important functions in tumor immunosurveillance, and in certain cases they can directly recognize HLA class II-expressing tumor cells. However, the underlying mechanism of intracellular Ag presentation to CD4(+) T cells by tumor cells has not yet been well characterized. We analyzed two naturally occurring human CD4(+) T cell lines specific for different peptides from cytosolic tumor Ag NY-ESO-1. Whereas both lines had the same HLA restriction and a similar ability to recognize exogenous NY-ESO-1 protein, only one CD4(+) T cell line recognized NY-ESO-1(+) HLA class II-expressing melanoma cells. Modulation of Ag processing in melanoma cells using specific molecular inhibitors and small interfering RNA revealed a previously undescribed peptide-selective Ag-presentation pathway by HLA class II(+) melanoma cells. The presentation required both proteasome and endosomal protease-dependent processing mechanisms, as well as cytosolic heat shock protein 90-mediated chaperoning. Such tumor-specific pathway of endogenous HLA class II Ag presentation is expected to play an important role in immunosurveillance or immunosuppression mediated by various subsets of CD4(+) T cells at the tumor local site. Furthermore, targeted activation of tumor-recognizing CD4(+) T cells by vaccination or adoptive transfer could be a suitable strategy for enhancing the efficacy of tumor immunotherapy.

Phenolic Compounds and Expression of 4CL Genes in Silver Birch Clones and Pt4CL1a Lines

PubMed Central

Sutela, Suvi; Hahl, Terhi; Tiimonen, Heidi; Aronen, Tuija; Ylioja, Tiina; Laakso, Tapio; Saranpää, Pekka; Chiang, Vincent; Julkunen-Tiitto, Riitta; Häggman, Hely

2014-01-01

A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins. PMID:25502441

The tetravalent organic cation spermine causes the gating of the IRK1 channel expressed in murine fibroblast cells.

PubMed Central

Ishihara, K; Hiraoka, M; Ochi, R

1996-01-01

1. The activation kinetics of the IRK1 channel stably expressed in L cells (a murine fibroblast cell line) were studied under the whole-cell voltage clamp. Without polyamines or Mg2+ in the pipettes, inward currents showed an exponential activation on hyperpolarization. The steep inward rectification of the currents around the reversal potential (Erev) could be described by the open-close transition of the channel with first-order kinetics. 2. When the tetravalent organic cation spermine (Spm) was added in the pipettes, the activation kinetics changed; this was explicable by the increase in the closing rate constant. The activation of the currents observed without Spm or Mg2+ in the pipettes was ascribed to the unblocking of the 'endogenous-Spm block'. 3. In the presence of the divalent cation putrescine (Put) or of Mg2+ in the pipettes, a different non-conductive state suppressed the outward currents on depolarization; the channels instantaneously changed to the open state on repolarization. As the depolarization was prolonged, this non-conductive state was replaced by the non-conductive state that shows an exponential activation on repolarization. This phenomenon was attributed to the redistribution of the channels from the Put- or Mg(2+)-blocked state to the 'endogenous Spm-blocked state' during depolarization. 4. In the presence of the trivalent cation spermidine (Spd) in the pipettes, two different non-conductive states occurred, showing a faster and a slower activation on repolarization. The rectification around Erev was mainly due to the non-conductive state showing a faster activation, which appeared to be the Spd-blocked state. During depolarization, redistribution of the channels to the 'endogenous Spm-blocked state' also occurred. 5. In the presence of Spd, Put or Mg2+ in the pipettes, the voltage dependence of the activation time constant reflecting the unblocking of the 'endogenous Spm' was shifted in the hyperpolarizing direction. 6. Our results suggest that the 'intrinsic gating' that shows the time-dependent activation on repolarization, and that is responsible for the inward rectification around Erev, reflects the blocking kinetics of the tetravalent Spm. PMID:8866861

The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells

PubMed Central

Gavina, Manuela; Za, Lorena; Molteni, Raffaella; Pardi, Ruggero; Curtis, Ivan de

2009-01-01

Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant-induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins. PMID:19912111

Interrogating the variation of element masses and distribution patterns in single cells using ICP-MS with a high efficiency cell introduction system.

PubMed

Wang, Hailong; Wang, Meng; Wang, Bing; Zheng, Lingna; Chen, Hanqing; Chai, Zhifang; Feng, Weiyue

2017-02-01

Cellular heterogeneity is an inherent condition of cell populations, which results from stochastic expression of genes, proteins, and metabolites. The heterogeneity of individual cells can dramatically influence cellular decision-making and cell fate. So far, our knowledge about how the variation of endogenous metals and non-metals in individual eukaryotic cells is limited. In this study, ICP-MS equipped with a high efficiency cell introduction system (HECIS) was developed as a method of single-cell ICP-MS (SC-ICP-MS). The method was applied to the single-cell analysis of Mn, Fe, Co, Cu, Zn, P, and S in human cancer cell lines (HeLa and A549) and normal human bronchial epithelial cell line (16HBE). The analysis showed obvious variation of the masses of Cu, Fe, Zn, and P in individual HeLa cells, and variation of Fe, Zn, and P in individual A549 cells. On the basis of the single-cell data, a multimodal distribution of the elements in the cell population was fitted, which showed marked differences among the various cell lines. Importantly, subpopulations of the elements were found in the cell populations, especially in the HeLa cancer cells. This study demonstrates that SC-ICP-MS is able to unravel the extent of variation of endogenous elements in individual cells, which will help to improve our fundamental understanding of cellular biology and reveal novel insights into human biology and medicine. Graphical abstract The variations of masses and distribution patterns of elements Mn, Fe, Co, Cu, Zn, P, and S in single cells were successfully detected by ICP-MS coupled with a high efficiency cell introduction system (HECIS).

TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.

PubMed

Ramalingam, Sivaprakash; Annaluru, Narayana; Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

2014-01-01

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences

NASA Astrophysics Data System (ADS)

Gong, Qianhong; Yu, Wengong; Dai, Jixun; Liu, Hongquan; Xu, Rifu; Guan, Huashi; Pan, Kehou

2007-01-01

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'-and 3'-flanking regions ( Tub5' and Tub3') up-and down-stream of β-glucuronidase (GUS) gene ( gusA), respectively, into pA, a derivative of pCAT®3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3'. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

High ADAM8 expression is associated with poor prognosis in patients with hepatocellular carcinoma.

PubMed

Zhang, Yun; Tan, Yong-Fei; Jiang, Chao; Zhang, Kai; Zha, Tian-Zhou; Zhang, Miao

2013-01-01

In this study,we investigated the ADAM8 expression in hepatocellular carcinoma (HCC) and its correlation with clinicopathologic features,including the survival of patients with HCC. Furthermore,we examined the biological processes regulated by ADAM8 during the development of using HepG2 cell line as a model system. We used immunohistochemistry to compare ADAM8 protein expression in HCC and normal liver tissues and further analyze the ADAM8 protein expression in clinicopathologically characterized 105 HCC cases.We stably knocked down the endogenous expression level of ADAM8 in HepG2 cells with specific shRNA-expressing lentiviral vector. Following the successful establishment of stable cells,we examined in vitro cell growth by MTT assay,anchorage-independent growth by soft-agar colony formation assay and cell migration/invasion by transwell and boyden chamber assay. And in addition,we also investigated the in vivo tumor growth by xenograft transplantation of HepG2 cells into nude mice. Protein expression level of ADAM8 was markedly higher in HCC tissues than that in the normal liver tissues (P = 0.0058).In addition,high expression of ADAM8 protein was positively correlated with serum AFP elevation,tumor size,histological differentiation,tumor recurrence,tumor metastasis,and tumor stage. Patients with higher ADAM8 expression showed a significantly shorter overall survival time than patients with low ADAM8 expression. Multivariate analysis suggested that ADAM8 expression might be an independent prognostic indicator (p = 0.016) for the survival of patients with HCC. ADAM8-specific shRNA (shADAM8) successfully knocked down its endogenous expression in HepG2 cells. Compared to the parental and control shRNA-transfected (shCtrl) HepG2 cells,the shADAM8 cells exhibited significantly reduced in vitro cell growth,anchorage-independent growth,cell migration and invasion (p < 0.05).In vivo,the xenograft transplants from shADAM8 cells gave rise to much smaller tumors as compared to those from shCtrl cells. High ADAM8 expression is associated with poor overall survival in patients with HCC. Down-regulation of ADAM8 inhibits the growth,anchorage-independent growth,migration and invasion of HepG2 cells. ADAM8 may be a potential target of antiangiogenic therapy for HCC.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity

PubMed Central

Care, Matthew A.; Cocco, Mario; Laye, Jon P.; Barnes, Nicholas; Huang, Yuanxue; Wang, Ming; Barrans, Sharon; Du, Ming; Jack, Andrew; Westhead, David R.; Doody, Gina M.; Tooze, Reuben M.

2014-01-01

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL. PMID:24875472

RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells

PubMed Central

2011-01-01

Background Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively. Methods We studied the use of down-regulation of this enzyme by RNA interference in three human cancer cell lines (A375, HeLa, and MCF-7) as a way to restore sensitivity to rADI in resistant cells. The expression of AS at levels of mRNA and protein was determined to understand the effect of RNA interference. Cell viability, cell cycle, and possible mechanism of the restore sensitivity of AS RNA interference in rADI treated cancer cells were evaluated. Results AS DNA was present in all cancer cell lines studied, however, the expression of this enzyme at the mRNA and protein level was different. In two rADI-resistant cell lines, one with endogenous AS expression (MCF-7 cells) and one with induced AS expression (HeLa cells), AS small interference RNA (siRNA) inhibited 37-46% of the expression of AS in MCF-7 cells. ASsiRNA did not affect cell viability in MCF-7 which may be due to the certain amount of residual AS protein. In contrast, ASsiRNA down-regulated almost all AS expression in HeLa cells and caused cell death after rADI treatment. Permanently down-regulated AS expression by short hairpin RNA (shRNA) made MCF-7 cells become sensitive to rADI via the inhibition of 4E-BP1-regulated mTOR signaling pathway. Conclusions Our results demonstrate that rADI-resistance can be altered via AS RNA interference. Although transient enzyme down-regulation (siRNA) did not affect cell viability in MCF-7 cells, permanent down-regulation (shRNA) overcame the problem of rADI-resistance due to the more efficiency in AS silencing. PMID:21453546

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

Lee, Seung-Hwan; Kim, Dong-Young; Jing, Feifeng

Developmental endothelial locus-1 (Del-1) is an endogenous anti-inflammatory molecule that is highly expressed in the lung and the brain and limits leukocyte migration to these tissues. We previously reported that the expression of Del-1 is positively regulated by p53 in lung endothelial cells. Although several reports have implicated the altered expression of Del-1 gene in cancer patients, little is known about its role in tumor cells. We here investigated the effect of Del-1 on the features of human lung carcinoma cells. Del-1 mRNA was found to be significantly decreased in the human lung adenocarcinoma cell lines A549 (containing wild typemore » of p53), H1299 (null for p53) and EKVX (mutant p53), compared to in human normal lung epithelial BEAS-2B cells and MRC-5 fibroblasts. The decrease of Del-1 expression was dependent on the p53 activity in the cell lines, but not on the expression of p53. Neither treatment with recombinant human Del-1 protein nor the introduction of adenovirus expressing Del-1 altered the expression of the apoptosis regulators BAX, PUMA and Bcl-2. Unexpectedly, the adenovirus-mediated overexpression of Del-1 gene into the lung carcinoma cell lines promoted proliferation and invasion of the lung carcinoma cells, as revealed by BrdU incorporation and transwell invasion assays, respectively. In addition, overexpression of the Del-1 gene enhanced features of epithelial–mesenchymal transition (EMT), such as increasing vimentin while decreasing E-cadherin in A549 cells, and increases in the level of Slug, an EMT-associated transcription regulator. Our findings demonstrated for the first time that there are deleterious effects of high levels of Del-1 in lung carcinoma cells, and suggest that Del-1 may be used as a diagnostic or prognostic marker for cancer progression, and as a novel therapeutic target for lung carcinoma. - Highlights: • Developmental Endothelial Locus-1 (Del-1) expression is downregulated in human lung cancer cells. • Overexpression of the Del-1 gene potentiates proliferation and invasion of lung carcinoma cells. • Del-1 may be used as a diagnostic or prognostic marker for lung cancer progression.« less

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene.

PubMed

Dale, Rodney M; Topczewski, Jacek

2011-09-15

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5' of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene

PubMed Central

Dale, Rodney M.; Topczewski, Jacek

2011-01-01

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5’ of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue. PMID:21723274

Interactions between Bmp-4 and Msx-1 act to restrict gene expression to odontogenic mesenchyme.

PubMed

Tucker, A S; Al Khamis, A; Sharpe, P T

1998-08-01

Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. Bmp4 has been identified as a candidate signalling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal (Vainio et al. [1993] Cell 75:45-58). A target gene for Bmp4 signalling is the homeobox gene Msx-1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx-1 expression. Msx-1 and Bmp-4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. In this study, we compare the temporal and spatial expression of these two genes to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. We show that changes in Bmp-4 expression precede changes in Msx-1 expression. At embryonic day (E)10.5-E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx-1 in underlying mesenchyme. At E11.5, Bmp-4 expression shifts from epithelium to mesenchyme and is rapidly followed by localised up-regulation of Msx-1 expression at the sites of Bmp-4 expression. Using cultured explants of developing mandibles, we confirm that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx-1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, we show that endogenous Msx-1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp-4 from the epithelium is responsible for regulating the early spatial expression of Msx-1. We also show that the mesenchymal shift in Bmp-4 is responsible for up-regulating Msx-1 specifically at the sites of future tooth formation. Thus, we establish that a reciprocal series of interactions act to restrict expression of both genes to future sites of tooth formation, creating a positive feedback loop that maintains expression of both genes in tooth mesenchymal cells.

Class I KNOX genes are associated with organogenesis during bulbil formation in Agave tequilana.

PubMed

Abraham-Juárez, María Jazmín; Martínez-Hernández, Aída; Leyva-González, Marco Antonio; Herrera-Estrella, Luis; Simpson, June

2010-09-01

Bulbil formation in Agave tequilana was analysed with the objective of understanding this phenomenon at the molecular and cellular levels. Bulbils formed 14-45 d after induction and were associated with rearrangements in tissue structure and accelerated cell multiplication. Changes at the cellular level during bulbil development were documented by histological analysis. In addition, several cDNA libraries produced from different stages of bulbil development were generated and partially sequenced. Sequence analysis led to the identification of candidate genes potentially involved in the initiation and development of bulbils in Agave, including two putative class I KNOX genes. Real-time reverse transcription-PCR and in situ hybridization revealed that expression of the putative Agave KNOXI genes occurs at bulbil initiation and specifically in tissue where meristems will develop. Functional analysis of Agave KNOXI genes in Arabidopsis thaliana showed the characteristic lobed phenotype of KNOXI ectopic expression in leaves, although a slightly different phenotype was observed for each of the two Agave genes. An Arabidopsis KNOXI (knat1) mutant line (CS30) was successfully complemented with one of the Agave KNOX genes and partially complemented by the other. Analysis of the expression of the endogenous Arabidopsis genes KNAT1, KNAT6, and AS1 in the transformed lines ectopically expressing or complemented by the Agave KNOX genes again showed different regulatory patterns for each Agave gene. These results show that Agave KNOX genes are functionally similar to class I KNOX genes and suggest that spatial and temporal control of their expression is essential during bulbil formation in A. tequilana.

Sequences required for induction of neurotensin receptor gene expression during neuronal differentiation of N1E-115 neuroblastoma cells.

PubMed

Tavares, D; Tully, K; Dobner, P R

1999-10-15

The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me(2)SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me(2)SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me(2)SO induction. The CACCC box is bound by at least one factor that is induced upon Me(2)SO treatment of N1E-115 cells. The Me(2)SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5' sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5'-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex.

Identification and characterization of two ankyrin-B isoforms in mammalian heart

PubMed Central

Wu, Henry C.; Yamankurt, Gokay; Luo, JiaLie; Subramaniam, Janani; Hashmi, Syed Shahrukh; Hu, Hongzhen; Cunha, Shane R.

2015-01-01

Aims Excitation–contraction coupling in cardiomyocytes requires the proper targeting and retention of membrane proteins to unique domains by adaptor proteins like ankyrin-B. While ankyrin-B has been shown to interact with a variety of membrane and structural proteins located at different subcellular domains in cardiomyocytes, what regulates the specificity of ankyrin-B for particular interacting proteins remains elusive. Methods and results Here, we report the identification of two novel ankyrin-B isoforms AnkB-188 and AnkB-212 in human, rat, and mouse hearts. Novel cDNAs for both isoforms were isolated by long-range PCR of reverse-transcribed mRNA isolated from human ventricular tissue. The isoforms can be discriminated based on their function and subcellular distribution in cardiomyocytes. Heterologous overexpression of AnkB-188 increases sodium–calcium exchanger (NCX) membrane expression and current, while selective knockdown of AnkB-188 in cardiomyocytes reduces NCX expression and localization in addition to causing irregular contraction rhythms. Using an isoform-specific antibody, we demonstrate that the expression of AnkB-212 is restricted to striated muscles and is localized to the M-line of cardiomyocytes by interacting with obscurin. Selective knockdown of AnkB-212 significantly attenuates the expression of endogenous ankyrin-B at the M-line but does not disrupt NCX expression at transverse tubules in cardiomyocytes. Conclusion The identification and characterization of two functionally distinct ankyrin-B isoforms in heart provide compelling evidence that alternative splicing of the ANK2 gene regulates the fidelity of ankyrin-B interactions with proteins. PMID:26109584

BMP6 down-regulates GDNF expression through SMAD1/5 and ERK1/2 signaling pathways in human granulosa-lutein cells.

PubMed

Zhang, Xin-Yue; Chang, Hsun-Ming; Taylor, Elizabeth L; Leung, Peter C K; Liu, Rui-Zhi

2018-05-09

Bone morphogenetic protein 6 (BMP6) is a critical regulator of follicular development that is expressed in mammalian oocytes and granulosa cells. Glial cell line-derived neurotrophic factor (GDNF) is an intraovarian neurotrophic factor that plays an essential role in regulating mammalian oocyte maturation. The aim of this study was to investigate the effect of BMP6 on the regulation of GDNF expression and the potential underlying mechanisms. We used an established immortalized human granulosa cell line (SVOG cells) and primary human granulosa-lutein cells as in vitro cell models. Our results showed that BMP6 significantly down-regulated the expression of GDNF in both SVOG and primary human granulosa-lutein cells. Using dual inhibition approaches (kinase receptor inhibitor and small interfering RNA knockdown), our results showed that both ALK2 and ALK3 are involved in BMP6-induced down-regulation of GDNF. In addition, BMP6 induced the phosphorylation of SMAD1/5/8 and ERK1/2 but not AKT or p38. Among three downstream mediators, both SMAD1 and SMAD5 are involved in BMP6-induced down-regulation of GDNF. Moreover, concomitant knockdown of endogenous SMAD4 and inhibition of ERK1/2 activity completely reversed BMP6-induced down-regulation of GDNF, indicating that both SMAD and ERK1/2 signaling pathways are required for the regulatory effect of BMP6 on GDNF expression. Our findings suggest an additional role for an intrafollicular growth factor in regulating follicular function through their paracrine interactions in human granulosa cells.

Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci

PubMed Central

Philippe, Claude; Vargas-Landin, Dulce B; Doucet, Aurélien J; van Essen, Dominic; Vera-Otarola, Jorge; Kuciak, Monika; Corbin, Antoine; Nigumann, Pilvi; Cristofari, Gaël

2016-01-01

LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants. DOI: http://dx.doi.org/10.7554/eLife.13926.001 PMID:27016617

Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line.

PubMed

Vassbotn, F S; Ostman, A; Langeland, N; Holmsen, H; Westermark, B; Heldin, C H; Nistér, M

1994-02-01

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF beta-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U-343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transformed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists.

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

Thiruselvam, Viswanathan; Sivaraman, Padavattan; Kumarevel, Thirumananseri, E-mail: kumarevel.thirumananseri@riken.jp

Highlights: • Crystal structure of ferritin was determined. • Endogenously expressed iron’s were identified. • Binuclear iron sites were observed at A and B active sites. - Abstract: Ferritin is an iron regulatory protein. It is responsible for storage and detoxification of excess iron thereby it regulates iron level in the body. Here we report the crystal structure of ferritin with two endogenously expressed Fe atoms binding in both the sites. The protein was purified and characterized by MALDI-TOF and N-terminal amino acid sequencing. The crystal belongs to I4 space group and it diffracted up to 2.5 Å. The structuralmore » analysis suggested that it crystallizes as hexamer and confirmed that it happened to be the first report of endogenously expressed Fe ions incorporated in both the A and B sites, situated in between the helices.« less

Differentially regulated gene expression in quiescence versus senescence and identification of ARID5A as a quiescence associated marker.

PubMed

Anwar, Tarique; Sen, Bijoya; Aggarwal, Savera; Nath, Rhisita; Pathak, Niteen; Katoch, Ajay; Aiyaz, Mohamed; Trehanpati, Nirupma; Khosla, Sanjeev; Ramakrishna, Gayatri

2018-05-01

In multicellular organisms majority of the cells remain in a non-dividing states of either quiescence (reversible) or senescence (irreversible). In the present study, gene expression signatures unique to quiescence and senescence were identified using microarray in osteosarcoma cell line, U2OS. It was noted that certain genes and pathways like NOD pathway was shared by both the growth arrest conditions. A major highlight of the present study was increased expression of number of chemokines and cytokines in both quiescence and senescence. While senescence-associated secretory phenotype (SASP) is well known, the quiescence-associated secretory phenotype (QASP) is relatively unknown and appeared novel in this study. ARID5A, a subunit of SWI/SNF complex was identified as a quiescence associated gene. The endogenous expression of ARID5A increased during serum starved condition of quiescence. Overexpression of ARID5A resulted in more number of cells in G0/G1 phase of cell cycle. Further ARID5A overexpressing cells when subjected to serum starvation showed a pronounced secretory phenotype. Overall, the present work has identified gene expression signatures which can distinguish quiescence from senescence. © 2017 Wiley Periodicals, Inc.

BAG3 promotes chondrosarcoma progression by upregulating the expression of β-catenin

PubMed Central

Shi, Huijuan; Chen, Wenfang; Dong, Yu; Lu, Xiaofang; Zhang, Wenhui; Wang, Liantang

2018-01-01

To investigate the roles of B-cell lymphoma-2 associated athanogene 3 (BAG3) in human chondrosarcoma and the potential mechanisms, the expression levels of BAG3 were detected in the present study, and the associations between BAG3 and clinical pathological parameters, clinical stage as well as the survival of patients were analyzed. The present study detected BAG3 mRNA and protein expression in the normal cartilage cell line HC-a and in SW1353 chondrosarcoma cells by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The BAG3 protein expression in 59 cases of chondrosarcoma, 30 patients with endogenous chondroma and 8 cases of normal cartilage was semi-quantitatively analyzed using the immunohistochemical method. In addition, the BAG3 protein expression level, the clinical pathological parameters, clinical stage and the survival time of patients with chondrosarcoma were analyzed. The plasmid transfection method was employed to upregulate the expression BAG3 and small RNA interference to downregulate the expression of BAG3 in SW1353 cells. The expression levels of BAG3 protein and mRNA were significantly increased in the chondrosarcoma cell line when compared with the normal cartilage cell line. The immunohistochemistry results indicated that BAG3 protein was overexpressed in the tissue of human chondrosarcoma. Statistical analysis showed that the expression level of BAG3 was significantly increased in the different Enneking staging of patients with chondrosarcoma and Tumor staging, and there were no statistical differences in age, gender, histological classification and tumor size. In the in vitro experiments, the data revealed that BAG3 significantly promoted chondrosarcoma cell proliferation, colony-formation, migration and invasion; however, it inhibited chondrosarcoma cell apoptosis. It was observed that BAG3 upregulated β-catenin expression at the mRNA and protein levels. In addition, BAG3 induced the expression of runt-related transcription factor 2 (RUNX2) in chondrosarcoma cells by upregulating β-catenin. These clinical analyses revealed a positive association between β-catenin and BAG3 in chondrosarcoma tumors. BAG3 was significantly increased in chondrosarcoma cells and tissues compared with the normal cartilage cells, tissue and cartilage benign tumors. Thus, BAG3 may serve as an oncogene in the development of chondrosarcoma via the induction of RUNX2 expression. The results of the present study contribute to further research on the biological development of chondrosarcoma. PMID:29484408

A gene trap transposon eliminates haematopoietic expression of zebrafish Gfi1aa, but does not interfere with haematopoiesis.

PubMed

Thambyrajah, Roshana; Ucanok, Deniz; Jalali, Maryam; Hough, Yasmin; Wilkinson, Robert Neil; McMahon, Kathryn; Moore, Chris; Gering, Martin

2016-09-01

A transposon-mediated gene trap screen identified the zebrafish line qmc551 that expresses a GFP reporter in primitive erythrocytes and also in haemogenic endothelial cells, which give rise to haematopoietic stem and progenitor cells (HSPCs) that seed sites of larval and adult haematopoiesis. The transposon that mediates this GFP expression is located in intron 1 of the gfi1aa gene, one of three zebrafish paralogs that encode transcriptional repressors homologous to mammalian Gfi1 and Gfi1b proteins. In qmc551 transgenics, GFP expression is under the control of the endogenous gfi1aa promoter, recapitulates early gfi1aa expression and allows live observation of gfi1aa promoter activity. While the transposon integration interferes with the expression of gfi1aa mRNA in haematopoietic cells, homozygous qmc551 fish are viable and fertile, and display normal primitive and definitive haematopoiesis. Retained expression of Gfi1b in primitive erythrocytes and up-regulation of Gfi1ab at the onset of definitive haematopoiesis in homozygous qmc551 carriers, are sufficient to allow normal haematopoiesis. This finding contradicts previously published morpholino data that suggested an essential role for zebrafish Gfi1aa in primitive erythropoiesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Overexpressed HDGF as an independent prognostic factor is involved in poor prognosis in Chinese patients with liver cancer

PubMed Central

2010-01-01

Background Hepatoma-derived growth factor (HDGF) is involved in the hepatocarcinogenesis. In this study, we investigated the HDGF expression in hepatocellular carcinoma (HCC) and its correlation with clinicopathologic features, including the survival of patients with HCC. Furthermore, we examined the biological processes regulated by HDGF during the development of using HepG2 cell line as a model system. Methods we used immunohistochemistry to compare HDGF protein expression in HCC and normal liver tissues and further analyze the HDGF protein expression in clinicopathologically characterized 137 HCC cases. We stably knocked down the endogenous expression level of HDGF in HepG2 cells with specific shRNA-expressing lentiviral vector. Following the successful establishment of stable cells, we examined in vitro cell growth by MTT assay, anchorage-independent growth by soft-agar colony formation assay and cell migration/invasion by transwell and boyden chamber assay. And in addition, we also investigated the in vivo tumor growth by xenograft transplantation of HepG2 cells into nude mice. Results Protein expression level of HDGF was markedly higher in HCC tissues than that in the normal liver tissues(P = 0.011). In addition, high expression of HDGF protein was positively correlated with T classification(p < 0.001), N classification (p < 0.001), and clinical stage (p < 0.001) of HCC patients. Patients with higher HDGF expression showed a significantly shorter overall survival time than did patients with low HDGF expression. Multivariate analysis suggested that HDGF expression might be an independent prognostic indicator(p < 0.001) for the survival of patients with HCC. HDGF-specific shRNA (shHDGF) successfully knocked down its endogenous expression in HepG2 cells. Compared to the parental and control shRNA-transfected (shCtrl) HepG2 cells, the shHDGF cells exhibited significantly reduced in vitro cell growth, anchorage-independent growth, cell migration and invasion (p < 0.05). In vivo, the xenograft transplants from shHDGF cells gave rise to much smaller tumors as compared to those from shCtrl cells. Conclusion High HDGF expression is associated with poor overall survival in patients with HCC. Down-regulation of HDGF inhibits the growth, anchorage-independent growth, migration and invasion of HepG2 cells. PMID:20846397

Validation of Endogenous Internal Real-Time PCR Controls in Renal Tissues

PubMed Central

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R.; Mrug, Michal

2009-01-01

Background Endogenous internal controls (‘reference’ or ‘housekeeping’ genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. Methods To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used ‘reference genes’ in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan® RT-PCR analyses and Affymetrix GeneChip® arrays, were normalized and tested for overall variance and equivalence of the means. Results Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. Conclusion A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. PMID:19729889

CD200 is induced by ERK and is a potential therapeutic target in melanoma

PubMed Central

Petermann, Kimberly B.; Rozenberg, Gabriela I.; Zedek, Daniel; Groben, Pamela; McKinnon, Karen; Buehler, Christin; Kim, William Y.; Shields, Janiel M.; Penland, Shannon; Bear, James E.; Thomas, Nancy E.; Serody, Jonathan S.; Sharpless, Norman E.

2007-01-01

Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM. PMID:18008004

Generation, Characterization and Application of Antibodies Directed against HERV-H Gag Protein in Colorectal Samples.

PubMed

Mullins, Christina S; Hühns, Maja; Krohn, Mathias; Peters, Sven; Cheynet, Valérie; Oriol, Guy; Guillotte, Michèle; Ducrot, Sandrine; Mallet, François; Linnebacher, Michael

2016-01-01

A substantial part of the human genome originates from transposable elements, remnants of ancient retroviral infections. Roughly 8% of the human genome consists of about 400,000 LTR elements including human endogenous retrovirus (HERV) sequences. Mainly, the interplay between epigenetic and post-transcriptional mechanisms is thought to silence HERV expression in most physiological contexts. Interestingly, aberrant reactivation of several HERV-H loci appears specific to colorectal carcinoma (CRC). The expression of HERV-H Gag proteins (Gag-H) was assessed using novel monoclonal mouse anti Gag-H antibodies. In a flow cytometry screen four antibody clones were tested on a panel of primary CRC cell lines and the most well performing ones were subsequently validated in western blot analysis. Finally, Gag-H protein expression was analyzed by immune histology on cell line cytospins and on clinical samples. There, we found a heterogeneous staining pattern with no background staining of endothelial, stromal and infiltrating immune cells but diffuse staining of the cytoplasm for positive tumor and normal crypt cells of the colonic epithelium. Taken together, the Gag-H antibody clone(s) present a valuable tool for staining of cells with colonic origin and thus form the basis for future more detailed investigations. The observed Gag-H protein staining in colonic epithelium crypt cells demands profound analyses of a potential role for Gag-H in the normal physiology of the human gut.

Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.

PubMed

Mabashi-Asazuma, Hideaki; Shi, Xianzong; Geisler, Christoph; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

2013-02-01

Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

Conjugated linoleic acid synthesis-related protein proteasome subunit α 5 (PSMA5) is increased by vaccenic acid treatment in goat mammary tissue.

PubMed

Jin, Y C; Li, Z H; Hong, Z S; Xu, C X; Han, J A; Choi, S H; Yin, J L; Zhang, Q K; Lee, K B; Kang, S K; Song, M K; Kim, Y J; Kang, H S; Choi, Y J; Lee, H G

2012-08-01

This study was conducted to identify proteins associated with the endogenous synthesis of conjugated linoleic acid (CLA) from trans-vaccenic acid (TVA; trans-11 C18:1, a precursor for CLA endogenous synthesis) in mammary tissues. Six lactating goats were divided into 2 groups. One group was given an intravenous bolus injection of TVA (150mg) twice daily over 4 d; the other group received saline injections. Treatment with TVA increased the concentration of cis-9,trans-11 CLA and TVA in goat milk. Additionally, TVA treatment increased the expression of stearoyl-CoA desaturase (SCD) in mammary tissue. Using 2-dimensional gel electrophoresis and electrospray ionization quadrupole time-of-flight mass spectrometry, 3 proteins affected by infusions of TVA were identified. Proteasome (prosome, macropain) subunit α type 5 (PSMA5) was upregulated, whereas peroxiredoxin-1 and translationally controlled tumor protein 1 were downregulated in TVA-treated animals compared with the vehicle-injected controls. Only the effect of TVA on PSMA5 could be confirmed by Western blot analysis. To further explore the regulation of PSMA5 in mammary epithelial cells when TVA is converted into CLA, we used a differentiated bovine mammary epithelial cell line treated with TVA for 6h. Changes in cis-9,trans-11 CLA concentrations and mRNA expression patterns of both SCD and PSMA5 were monitored. The concentration of cis-9,trans-11 CLA increased after TVA treatment. The mRNA expression level of PSMA5 was significantly elevated to 6h, but SCD mRNA expression only increased in 2h after TVA treatment. These results indicate that PSMA5 is highly expressed in goat mammary tissue and bovine mammary epithelial cells when TVA is converted into CLA. Our data suggest that PSMA5 protein is associated with CLA biosynthesis in mammary tissue. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

C-terminal cleavage of DeltaNp63alpha is associated with TSA-induced apoptosis in immortalized corneal epithelial cells.

PubMed

Robertson, Danielle M; Ho, Su-Inn; Cavanagh, H Dwight

2010-08-01

In the central human corneal epithelium, loss of DeltaNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for DeltaNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for DeltaNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual DeltaNp63 isoforms, DeltaNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 muM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous DeltaNp63alpha, DeltaNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. Transient expression of DeltaNp63-EGFP alpha and beta isoforms resulted in the formation of a smaller isoform similar in size to DeltaNp63gamma-EGFP. FRAP demonstrated that DeltaNp63alpha-EGFP has greater immobile fraction than beta or gamma. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous DeltaNp63alpha and p53. TSA upregulated DeltaNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of DeltaNp63alpha-EGFP. siRNA knockdown of DeltaNp63alpha correlated with a reduction in p53 independently of TSA. DeltaNp63alpha is the dominant active isoform in corneal epithelial cell nuclei. Loss of DeltaNp63alpha occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.

Processed pseudogenes of human endogenous retroviruses generated by LINEs: their integration, stability, and distribution.

PubMed

Pavlícek, Adam; Paces, Jan; Elleder, Daniel; Hejnar, Jirí

2002-03-01

We report here the presence of numerous processed pseudogenes derived from the W family of endogenous retroviruses in the human genome. These pseudogenes are structurally colinear with the retroviral mRNA followed by a poly(A) tail. Our analysis of insertion sites of HERV-W processed pseudogenes shows a strong preference for the insertion motif of long interspersed nuclear element (LINE) retrotransposons. The genomic distribution, stability during evolution, and frequent truncations at the 5' end resemble those of the pseudogenes generated by LINEs. We therefore suggest that HERV-W processed pseudogenes arose by multiple and independent LINE-mediated retrotransposition of retroviral mRNA. These data document that the majority of HERV-W copies are actually nontranscribed promoterless pseudogenes. The current search for HERV-Ws associated with several human diseases should concentrate on a small subset of transcriptionally competent elements.

Identification and overexpression of a knotted1-like transcription factor in switchgrass ( Panicum virgatum L.) for lignocellulosic feedstock improvement

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

Wuddineh, Wegi A.; Mazarei, Mitra; Zhang, Ji -Yi

High biomass production and wide adaptation has made switchgrass ( Panicum virgatum L.) an important candidate lignocellulosic bioenergy crop. One major limitation of this and other lignocellulosic feedstocks is the recalcitrance of complex carbohydrates to hydrolysis for conversion to biofuels. Lignin is the major contributor to recalcitrance as it limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars. Therefore, genetic manipulation of the lignin biosynthesis pathway is one strategy to reduce recalcitrance. Here, we identified a switchgrass Knotted1 transcription factor, PvKN1, with the aim of genetically engineering switchgrass for reduced biomass recalcitrance for biofuel production. Genemore » expression of the endogenous PvKN1 gene was observed to be highest in young inflorescences and stems. Ectopic overexpression of PvKN1 in switchgrass altered growth, especially in early developmental stages. Transgenic lines had reduced expression of most lignin biosynthetic genes accompanied by a reduction in lignin content suggesting the involvement of PvKN1 in the broad regulation of the lignin biosynthesis pathway. Moreover, the reduced expression of the Gibberellin 20-oxidase (GA20ox) gene in tandem with the increased expression of Gibberellin 2-oxidase (GA2ox) genes in transgenic PvKN1 lines suggest that PvKN1 may exert regulatory effects via modulation of GA signaling. Furthermore, overexpression of PvKN1 altered the expression of cellulose and hemicellulose biosynthetic genes and increased sugar release efficiency in transgenic lines. Our findings demonstrated that switchgrass PvKN1 is a putative ortholog of maize KN1 that is linked to plant lignification and cell wall and development traits as a major regulatory gene. Therefore, targeted overexpression of PvKN1 in bioenergy feedstocks may provide one feasible strategy for reducing biomass recalcitrance and simultaneously improving plant growth characteristics.« less

Identification and overexpression of a knotted1-like transcription factor in switchgrass ( Panicum virgatum L.) for lignocellulosic feedstock improvement

DOE PAGES

Wuddineh, Wegi A.; Mazarei, Mitra; Zhang, Ji -Yi; ...

2016-04-28

High biomass production and wide adaptation has made switchgrass ( Panicum virgatum L.) an important candidate lignocellulosic bioenergy crop. One major limitation of this and other lignocellulosic feedstocks is the recalcitrance of complex carbohydrates to hydrolysis for conversion to biofuels. Lignin is the major contributor to recalcitrance as it limits the accessibility of cell wall carbohydrates to enzymatic breakdown into fermentable sugars. Therefore, genetic manipulation of the lignin biosynthesis pathway is one strategy to reduce recalcitrance. Here, we identified a switchgrass Knotted1 transcription factor, PvKN1, with the aim of genetically engineering switchgrass for reduced biomass recalcitrance for biofuel production. Genemore » expression of the endogenous PvKN1 gene was observed to be highest in young inflorescences and stems. Ectopic overexpression of PvKN1 in switchgrass altered growth, especially in early developmental stages. Transgenic lines had reduced expression of most lignin biosynthetic genes accompanied by a reduction in lignin content suggesting the involvement of PvKN1 in the broad regulation of the lignin biosynthesis pathway. Moreover, the reduced expression of the Gibberellin 20-oxidase (GA20ox) gene in tandem with the increased expression of Gibberellin 2-oxidase (GA2ox) genes in transgenic PvKN1 lines suggest that PvKN1 may exert regulatory effects via modulation of GA signaling. Furthermore, overexpression of PvKN1 altered the expression of cellulose and hemicellulose biosynthetic genes and increased sugar release efficiency in transgenic lines. Our findings demonstrated that switchgrass PvKN1 is a putative ortholog of maize KN1 that is linked to plant lignification and cell wall and development traits as a major regulatory gene. Therefore, targeted overexpression of PvKN1 in bioenergy feedstocks may provide one feasible strategy for reducing biomass recalcitrance and simultaneously improving plant growth characteristics.« less

The APETALA-2-like transcription factor OsAP2-39 controls key interactions between abscisic acid and gibberellin in rice.

PubMed

Yaish, Mahmoud W; El-Kereamy, Ashraf; Zhu, Tong; Beatty, Perrin H; Good, Allen G; Bi, Yong-Mei; Rothstein, Steven J

2010-09-09

The interaction between phytohormones is an important mechanism which controls growth and developmental processes in plants. Deciphering these interactions is a crucial step in helping to develop crops with enhanced yield and resistance to environmental stresses. Controlling the expression level of OsAP2-39 which includes an APETALA 2 (AP2) domain leads to phenotypic changes in rice. Overexpression of OsAP2-39 leads to a reduction in yield by decreasing the biomass and the number of seeds in the transgenic rice lines. Global transcriptome analysis of the OsAP2-39 overexpression transgenic rice revealed the upregulation of a key abscisic acid (ABA) biosynthetic gene OsNCED-I which codes for 9-cis-epoxycarotenoid dioxygenase and leads to an increase in the endogenous ABA level. In addition to OsNCED-1, the gene expression analysis revealed the upregulation of a gene that codes for the Elongation of Upper most Internode (EUI) protein, an enzyme that catalyzes 16α, 17-epoxidation of non-13-hydroxylated GAs, which has been shown to deactivate gibberellins (GAs) in rice. The exogenous application of GA restores the wild-type phenotype in the transgenic line and ABA application induces the expression of EUI and suppresses the expression of OsAP2-39 in the wild-type line. These observations clarify the antagonistic relationship between ABA and GA and illustrate a mechanism that leads to homeostasis of these hormones. In vivo and in vitro analysis showed that the expression of both OsNCED-1 and EUI are directly controlled by OsAP2-39. Together, these results reveal a novel mechanism for the control of the ABA/GA balance in rice which is regulated by OsAP2-39 that in turn regulates plant growth and seed production.

The APETALA-2-Like Transcription Factor OsAP2-39 Controls Key Interactions between Abscisic Acid and Gibberellin in Rice

PubMed Central

Yaish, Mahmoud W.; El-kereamy, Ashraf; Zhu, Tong; Beatty, Perrin H.; Good, Allen G.; Bi, Yong-Mei; Rothstein, Steven J.

2010-01-01

The interaction between phytohormones is an important mechanism which controls growth and developmental processes in plants. Deciphering these interactions is a crucial step in helping to develop crops with enhanced yield and resistance to environmental stresses. Controlling the expression level of OsAP2-39 which includes an APETALA 2 (AP2) domain leads to phenotypic changes in rice. Overexpression of OsAP2-39 leads to a reduction in yield by decreasing the biomass and the number of seeds in the transgenic rice lines. Global transcriptome analysis of the OsAP2-39 overexpression transgenic rice revealed the upregulation of a key Abscisic Acid (ABA) biosynthetic gene OsNCED-I which codes for 9-cis-epoxycarotenoid dioxygenase and leads to an increase in the endogenous ABA level. In addition to OsNCED-1, the gene expression analysis revealed the upregulation of a gene that codes for the Elongation of Upper most Internode (EUI) protein, an enzyme that catalyzes 16α, 17-epoxidation of non-13-hydroxylated GAs, which has been shown to deactivate gibberellins (GAs) in rice. The exogenous application of GA restores the wild-type phenotype in the transgenic line and ABA application induces the expression of EUI and suppresses the expression of OsAP2-39 in the wild-type line. These observations clarify the antagonistic relationship between ABA and GA and illustrate a mechanism that leads to homeostasis of these hormones. In vivo and in vitro analysis showed that the expression of both OsNCED-1 and EUI are directly controlled by OsAP2-39. Together, these results reveal a novel mechanism for the control of the ABA/GA balance in rice which is regulated by OsAP2-39 that in turn regulates plant growth and seed production. PMID:20838584

Expression of phytoene synthase1 and carotene desaturase crtI genes result in an increase in the total carotenoids content in transgenic elite wheat (Triticum aestivum L.).

PubMed

Cong, Ling; Wang, Cheng; Chen, Ling; Liu, Huijuan; Yang, Guangxiao; He, Guangyuan

2009-09-23

Dietary micronutrient deficiencies, such as the lack of vitamin A, are a major source of morbidity and mortality worldwide. Carotenoids in food can function as provitamin A in humans, while grains of Chinese elite wheat cultivars generally have low carotenoid contents. To increase the carotenoid contents in common wheat endosperm, transgenic wheat has been generated by expressing the maize y1 gene encoding phytoene synthase driven by a endosperm-specific 1Dx5 promoter in the elite wheat (Triticum aestivum L.) variety EM12, together with the bacterial phytoene desaturase crtI gene from Erwinia uredovora under the constitutive CaMV 35S promoter control. A clear increase of the carotenoid content was detected in the endosperms of transgenic wheat that visually showed a light yellow color. The total carotenoids content was increased up to 10.8-fold as compared with the nontransgenic EM12 cultivar. To test whether the variability of total carotenoid content in different transgenic lines was due to differences in the transgene copy number or expression pattern, Southern hybridization and semiquantitative reverse transcriptase polymerase chain reaction analyses were curried out. The results showed that transgene copy numbers and transcript levels did not associate well with carotenoid contents. The expression patterns of endogenous carotenoid genes, such as the phytoene synthases and carotene desaturases, were also investigated in wild-type and transgenic wheat lines. No significant changes in expression levels of these genes were detected in the transgenic endosperms, indicating that the increase in carotenoid transgenic wheat endosperms resulted from the expression of transgenes.

S100A8/A9 regulates MMP-2 expression and invasion and migration by carcinoma cells.

PubMed

Silva, Emmanuel J; Argyris, Prokopios P; Zou, Xianqiong; Ross, Karen F; Herzberg, Mark C

2014-10-01

Intracellular calprotectin (S100A8/A9) functions in the control of the cell cycle checkpoint at G2/M. Dysregulation of S100A8/A9 appears to cause loss of the checkpoint, which frequently characterizes head and neck squamous cell carcinoma (HNSCC). In the present study, we analyzed carcinoma cells for other S100A8/A9-directed changes in malignant phenotype. Using a S100A8/A9-negative human carcinoma cell line (KB), transfection to express S100A8 and S100A9 caused selective down-regulation of MMP-2 and inhibited in vitro invasion and migration. Conversely, silencing of endogenous S100A8 and S100A9 expression in TR146 cells, a well-differentiated HNSCC cell line, increased MMP-2 activity and in vitro invasion and migration. When MMP-2 expression was silenced, cells appeared to assume a less malignant phenotype. To more closely model the architecture of cell growth in vivo, cells were grown in a 3D collagen substrate, which was compared to 2D. Growth on 3D substrates caused greater MMP-2 expression. Whereas hypermethylation of CpG islands occurs frequently in HNSCC, S100A8/A9-dependent regulation of MMP-2 could not be explained by modification of the upstream promoters of MMP2 or TIMP2. Collectively, these results suggest that intracellular S100A8/A9 contributes to the cancer cell phenotype by modulating MMP-2 expression and activity to regulate cell migration and mobility. Published by Elsevier Ltd.

lncRNA CCAT1 promotes cell proliferation, migration, and invasion by down-regulation of miR-143 in FTC-133 thyroid carcinoma cell line.

PubMed

Yang, Tianzheng; Zhai, Hongyan; Yan, Ruihong; Zhou, Zhenhu; Gao, Lei; Wang, Luqing

2018-01-01

Thyroid cancer is a common malignant tumor. Long non-coding RNA colon cancer-associated transcript 1 (lncRNA CCAT1) is highly expressed in many cancers; however, the molecular mechanism of CCAT1 in thyroid cancer remains unclear. Hence, this study aimed to investigate the effect of CCAT1 on human thyroid cancer cell line FTC-133. FTC-133 cells were transfected with CCAT1 expressing vector, CCAT1 shRNA, miR-143 mimic, and miR-143 inhibitor, respectively. After different treatments, cell viability, proliferation, migration, invasion, and apoptosis were measured. Moreover, the regulatory relationship of CCAT1 and miR-143, as well as miR-143 and VEGF were tested using dual-luciferase reporter assay. The relative expressions of CCAT1, miR-143, and VEGF were tested by qRT-PCR. The expressions of apoptosis-related factors and corresponding proteins in PI3K/AKT and MAPK pathways were analyzed using western blot analysis. The results suggested that CCAT1 was up-regulated in the FTC-133 cells. CCAT1 suppression decreased FTC-133 cell viability, proliferation, migration, invasion, and miR-143 expression, while it increased apoptosis and VEGF expression. CCAT1 might act as a competing endogenous RNA (ceRNA) for miR-143. Moreover, CCAT1 activated PI3K/AKT and MAPK signaling pathways through inhibition of miR-143. This study demonstrated that CCAT1 exhibited pro-proliferative and pro-metastasis functions on FTC-133 cells and activated PI3K/AKT and MAPK signaling pathways via down-regulation of miR-143. These findings will provide a possible target for clinical treatment of thyroid cancer.

Loss of p53-inducible long non-coding RNA LINC01021 increases chemosensitivity

PubMed Central

Kaller, Markus; Götz, Ursula; Hermeking, Heiko

2017-01-01

We have previously identified the long non-coding RNA LINC01021 as a direct p53 target (Hünten et al. Mol Cell Proteomics. 2015; 14:2609-2629). Here, we show that LINC01021 is up-regulated in colorectal cancer (CRC) cell lines upon various p53-activating treatments. The LINC01021 promoter and the p53 binding site lie within a MER61C LTR, which originated from insertion of endogenous retrovirus 1 (ERV1) sequences. Deletion of this MER61C element by a CRISPR/Cas9 approach, as well as siRNA-mediated knockdown of LINC01021 RNA significantly enhanced the sensitivity of the CRC cell line HCT116 towards the chemotherapeutic drugs doxorubicin and 5-FU, suggesting that LINC01021 is an integral part of the p53-mediated response to DNA damage. Inactivation of LINC01021 and also its ectopic expression did not affect p53 protein expression and transcriptional activity, implying that LINC01021 does not feedback to p53. Furthermore, in CRC patient samples LINC01021 expression positively correlated with a wild-type p53-associated gene expression signature. LINC01021 expression was increased in primary colorectal tumors and displayed a bimodal distribution that was particularly pronounced in the mesenchymal CMS4 consensus molecular subtype of CRCs. CMS4 tumors with low LINC01021 expression were associated with poor patient survival. Our results suggest that the genomic redistribution of ERV1-derived p53 response elements and generation of novel p53-inducible lncRNA-encoding genes was selected for during primate evolution as integral part of the cellular response to various forms of genotoxic stress. PMID:29262524

G-Protein-Coupled Receptor Gpr17 Expression in Two Multiple Sclerosis Remyelination Models.

PubMed

Nyamoya, Stella; Leopold, Patrizia; Becker, Birte; Beyer, Cordian; Hustadt, Fabian; Schmitz, Christoph; Michel, Anne; Kipp, Markus

2018-06-05

In multiple sclerosis patients, demyelination is prominent in both the white and gray matter. Chronic clinical deficits are known to result from acute or chronic injury to the myelin sheath and inadequate remyelination. The underlying molecular mechanisms of remyelination and its failure remain currently unclear. Recent studies have recognized G protein-coupled receptor 17 (GPR17) as an important regulator of oligodendrocyte development and remyelination. So far, the relevance of GPR17 for myelin repair was mainly tested in remyelinating white matter lesions. The relevance of GPR17 for gray matter remyelination as well as remyelination of chronic white matter lesions was not addressed so far. Here, we provide a detailed characterization of GPR17 expression during experimental de- and remyelination. Experimental lesions with robust and limited endogenous remyelination capacity were established by either acute or chronic cuprizone-induced demyelination. Furthermore, remyelinating lesions were induced by the focal injection of lysophosphatidylcholine (LPC) into the corpus callosum. GPR17 expression was analyzed by complementary techniques including immunohistochemistry, in situ hybridization, and real-time PCR. In control animals, GPR17 + cells were evenly distributed in the corpus callosum and cortex and displayed a highly ramified morphology. Virtually all GPR17 + cells also expressed the oligodendrocyte-specific transcription factor OLIG2. After acute cuprizone-induced demyelination, robust endogenous remyelination was evident in the white matter corpus callosum but not in the gray matter cortex. Endogenous callosal remyelination was paralleled by a robust induction of GPR17 expression which was absent in the gray matter cortex. Higher numbers of GPR17 + cells were as well observed after LPC-induced focal white matter demyelination. In contrast, densities of GPR17 + cells were comparable to control animals after chronic cuprizone-induced demyelination indicating quiescence of this cell population. Our findings demonstrate that GPR17 expression induction correlates with acute demyelination and sufficient endogenous remyelination. This strengthens the view that manipulation of this receptor might be a therapeutic opportunity to support endogenous remyelination.

Characterization of Soybean Genetically Modified for Drought Tolerance in Field Conditions

PubMed Central

Fuganti-Pagliarini, Renata; Ferreira, Leonardo C.; Rodrigues, Fabiana A.; Molinari, Hugo B. C.; Marin, Silvana R. R.; Molinari, Mayla D. C.; Marcolino-Gomes, Juliana; Mertz-Henning, Liliane M.; Farias, José R. B.; de Oliveira, Maria C. N.; Neumaier, Norman; Kanamori, Norihito; Fujita, Yasunari; Mizoi, Junya; Nakashima, Kazuo; Yamaguchi-Shinozaki, Kazuko; Nepomuceno, Alexandre L.

2017-01-01

Drought is one of the most stressful environmental factor causing yield and economic losses in many soybean-producing regions. In the last decades, transcription factors (TFs) are being used to develop genetically modified plants more tolerant to abiotic stresses. Dehydration responsive element binding (DREB) and ABA-responsive element-binding (AREB) TFs were introduced in soybean showing improved drought tolerance, under controlled conditions. However, these results may not be representative of the way in which plants behave over the entire season in the real field situation. Thus, the objectives of this study were to analyze agronomical traits and physiological parameters of AtDREB1A (1Ab58), AtDREB2CA (1Bb2193), and AtAREB1 (1Ea2939) GM lines under irrigated (IRR) and non-irrigated (NIRR) conditions in a field experiment, over two crop seasons and quantify transgene and drought-responsive genes expression. Results from season 2013/2014 revealed that line 1Ea2939 showed higher intrinsic water use and leaf area index. Lines 1Ab58 and 1Bb2193 showed a similar behavior to wild-type plants in relation to chlorophyll content. Oil and protein contents were not affected in transgenic lines in NIRR conditions. Lodging, due to plentiful rain, impaired yield from the 1Ea2939 line in IRR conditions. qPCR results confirmed the expression of the inserted TFs and drought-responsive endogenous genes. No differences were identified in the field experiment performed in crop season 2014/2015, probably due to the optimum rainfall volume during the cycle. These field screenings showed promising results for drought tolerance. However, additional studies are needed in further crop seasons and other sites to better characterize how these plants may outperform the WT under field water deficit. PMID:28443101

Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization.

PubMed

Roberts, Brock; Haupt, Amanda; Tucker, Andrew; Grancharova, Tanya; Arakaki, Joy; Fuqua, Margaret A; Nelson, Angelique; Hookway, Caroline; Ludmann, Susan A; Mueller, Irina A; Yang, Ruian; Horwitz, Rick; Rafelski, Susanne M; Gunawardane, Ruwanthi N

2017-10-15

We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in <0.1-4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line-generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. © 2017 Roberts, Haupt, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.

PubMed

Chen, S; Blanck, G; Pollack, R E

1983-09-01

We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation.

Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.

PubMed Central

Chen, S; Blanck, G; Pollack, R E

1983-01-01

We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation. Images PMID:6310588

Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction

PubMed Central

Malliaras, Konstantinos; Ibrahim, Ahmed; Tseliou, Eleni; Liu, Weixin; Sun, Baiming; Middleton, Ryan C; Seinfeld, Jeffrey; Wang, Lai; Sharifi, Behrooz G; Marbán, Eduardo

2014-01-01

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium. PMID:24797668

Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

NASA Astrophysics Data System (ADS)

Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jimmy

2015-01-01

Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1 and OPR) in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

Glutamine synthetase gene knockout-human embryonic kidney 293E cells for stable production of monoclonal antibodies.

PubMed

Yu, Da Young; Lee, Sang Yoon; Lee, Gyun Min

2018-05-01

Previously, it was inferred that a high glutamine synthetase (GS) activity in human embryonic kidney (HEK) 293E cells results in elevated resistance to methionine sulfoximine (MSX) and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. © 2018 Wiley Periodicals, Inc.

Identification of Transposable Elements Contributing to Tissue-Specific Expression of Long Non-Coding RNAs

PubMed Central

Chishima, Takafumi; Iwakiri, Junichi

2018-01-01

It has been recently suggested that transposable elements (TEs) are re-used as functional elements of long non-coding RNAs (lncRNAs). This is supported by some examples such as the human endogenous retrovirus subfamily H (HERVH) elements contained within lncRNAs and expressed specifically in human embryonic stem cells (hESCs), as required to maintain hESC identity. There are at least two unanswered questions about all lncRNAs. How many TEs are re-used within lncRNAs? Are there any other TEs that affect tissue specificity of lncRNA expression? To answer these questions, we comprehensively identify TEs that are significantly related to tissue-specific expression levels of lncRNAs. We downloaded lncRNA expression data corresponding to normal human tissue from the Expression Atlas and transformed the data into tissue specificity estimates. Then, Fisher’s exact tests were performed to verify whether the presence or absence of TE-derived sequences influences the tissue specificity of lncRNA expression. Many TE–tissue pairs associated with tissue-specific expression of lncRNAs were detected, indicating that multiple TE families can be re-used as functional domains or regulatory sequences of lncRNAs. In particular, we found that the antisense promoter region of L1PA2, a LINE-1 subfamily, appears to act as a promoter for lncRNAs with placenta-specific expression. PMID:29315213

Cucumber (Cucumis sativus L.) Nitric Oxide Synthase Associated Gene1 (CsNOA1) Plays a Role in Chilling Stress

PubMed Central

Liu, Xingwang; Liu, Bin; Xue, Shudan; Cai, Yanlinq; Qi, Wenzhu; Jian, Chen; Xu, Shuo; Wang, Ting; Ren, Huazhong

2016-01-01

Nitric oxide (NO) is a gaseous signaling molecule in plants, transducing information as a result of exposure to low temperatures. However, the underlying molecular mechanism linking NO with chilling stress is not well understood. Here, we functionally characterized the cucumber (Cucumis sativus L.) nitric oxide synthase-associated gene, NITRIC OXIDE ASSOCIATED 1 (CsNOA1). Expression analysis of CsNOA1, using quantitative real-time PCR, in situ hybridization, and a promoter::β-glucuronidase (GUS) reporter assay, revealed that it is expressed mainly in the root and shoot apical meristem (SAM), and that expression is up-regulated by low temperatures. A CsNOA1-GFP fusion protein was found to be localized in the mitochondria, and ectopic expression of CsNOA1 in the A. thaliana noa1 mutant partially rescued the normal phenotype. When overexpressing CsNOA1 in the Atnoa1 mutant under normal condition, no obvious phenotypic differences was observed between its wild type and transgenic plants. However, the leaves from mutant plant grown under chilling conditions showed hydrophanous spots and wilting. Physiology tolerance markers, chlorophyll fluorescence parameter (Fv/Fm), and electrolyte leakage, were observed to dramatically change, compared mutant to overexpressing lines. Transgenic cucumber plants revealed that the gene is required by seedlings to tolerate chilling stress: constitutive over-expression of CsNOA1 led to a greater accumulation of soluble sugars, starch, and an up-regulation of Cold-regulatory C-repeat binding factor3 (CBF3) expression as well as a lower chilling damage index (CI). Conversely, suppression of CsNOA1 expression resulted in the opposite phenotype and a reduced NO content compared to wild type plants. Those results suggest that CsNOA1 regulates cucumber seedlings chilling tolerance. Additionally, under normal condition, we took several classic inhibitors to perform, and detect endogenous NO levels in wild type cucumber seedling. The results suggest that generation of endogenous NO in cucumber leaves occurs largely independently in the (CsNOA1) and nitrate reductase (NR) pathway. PMID:27891134

Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors.

PubMed

Galli, Uwe M; Sauter, Marlies; Lecher, Bernd; Maurer, Simone; Herbst, Hermann; Roemer, Klaus; Mueller-Lantzsch, Nikolaus

2005-04-28

Germ cell tumors (GCTs) are among the most common malignancies in young men. We have previously documented that patients with GCT frequently produce serum antibodies directed against proteins encoded by human endogenous retrovirus (HERV) type K sequences. Transcripts originating from the env gene of HERV-K, including the rec-relative of human immunodeficiency virus rev, are highly expressed in GCTs. We report here that mice that inducibly express HERV-K rec show a disturbed germ cell development and may exhibit, by 19 months of age, changes reminiscent of carcinoma in situ, the predecessor lesion of classic seminoma in humans. This provides the first direct evidence that the expression of a human endogenous retroviral gene previously established as a marker in human germ cell tumors may contribute to organ-specific tumorigenesis in a transgenic mouse model.

Upregulation of Endogenous HMOX1 Expression by a Computer-Designed Artificial Transcription Factor

PubMed Central

Guo, Hongfeng; Tian, Yi; Lu, Hai; Wei, Yong; Ying, Dajun

2010-01-01

Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases. PMID:20706680

The Naïve Murine Cornea as a Model System to Identify Novel Endogenous Regulators of Lymphangiogenesis: TRAIL and rtPA.

PubMed

Regenfuß, Birgit; Dreisow, Marie-Luise; Hos, Deniz; Masli, Sharmila; Bock, Felix; Cursiefen, Claus

2015-06-01

In the murine cornea, which is an established model for analyzing pathologic lymphatic vessel growth, phenotypic heterogeneity of the endogenous lymphatic vessels in the limbus of the cornea was previously described. In this study, the cornea of BALB/c, C57BL/6, and FVB mice with different limbal lymphangiogenic phenotypes was analyzed to identify novel candidates potentially influencing lymphatic vessel growth. Pathway specific expression analysis of the cornea was performed to identify novel candidate genes. Corneal protein expression of the respective candidates was analyzed by fluorescent immunohistochemistry. The effect of the candidates on proliferation of human dermal lymphatic endothelial cells (HDLECs) was analyzed by BrdU proliferation ELISA. Thirteen genes were differentially regulated in corneas of mouse strains with more endogenous limbal lymphatic vessels (high-lymphangiogenic) (C57BL/6) compared to mouse strains with less endogenous limbal lymphatic vessels (low-lymphangiogenic) (BALB/c, FVB). Two candidates, Tumor necrosis factor (ligand) superfamily member 10 (Tnfsf10/Trail) and Plasminogen activator, tissue (Plat/tPA) were expressed in the cornea of BALB/c and C57BL/6 mice on the protein level. In vitro, Trail and recombinant tPA inhibited the proliferation of human dermal lymphatic endothelial cells. Molecular analysis of the naive cornea in mouse strains with different limbal lymphatic phenotypes is a valuable model to identify novel endogenous regulators of lymphangiogenesis.

PIK3C2A mRNA functions as a miR-124 sponge to facilitate CD151 expression and enhance malignancy of hepatocellular carcinoma cells.

PubMed

Liu, Tao; Zu, Cai-Hua; Wang, Shu-Sen; Song, Hong-Li; Wang, Zheng-Lu; Xu, Xin-Nv; Liu, Hong-Sheng; Wang, Yu-Liang; Shen, Zhong-Yang

2016-07-12

Competing endogenous RNAs (ceRNAs) are RNA transcripts that can crosstalk with each other by competing for shared microRNAs (miRNAs) through miRNA response elements (MREs). Involved in ceRNA networks, the RNA transcripts may be in a balance, disruption of which could lead to tumorigenesis. Here we reveal a ceRNA interaction between PIK3C2A and CD151 mRNAs in hepatocellular carcinoma (HCC) cells. PIK3C2A is a candidate ceRNA of CD151 because mRNA 3' untranslated regions (3'UTRs) of these two genes contain miR-124 binding sites. miR-124 is downregulated, while PIK3C2A and CD151 are upregulated in HCC cells compared with normal hepatocytes. Direct and negative regulation of PIK3C2A and CD151 by miR-124 was confirmed in HCC cells. miR-124 and the two potential ceRNAs are all recruited to the RNA-induced silencing complex (RISC). In HCC cell lines QGY- 7703 and SMMC-7721, and normal hepatic cell line HL-7702, miR-124 plays a tumor suppressor role by targeting PIK3C2A and CD151. The MREs within PIK3C2A 3'UTR can independently stimulate CD151 expression level by acting as miR-124 decoys. PIK3C2A MREs enhance HCC cell malignancy by absorbing endogenous miR-124 and activating CD151 in HCC cells. We conclude that PIK3C2A 3'UTR functions as a trans activator to stimulate CD151 by competing for miR-124 binding in HCC cells. The collaboration of PIK3C2A and CD151 through ceRNA mechanism may be implicated in HCC initiation and development.

Late-onset cone photoreceptor degeneration induced by R172W mutation in Rds and partial rescue by gene supplementation.

PubMed

Conley, Shannon; Nour, May; Fliesler, Steven J; Naash, Muna I

2007-12-01

R172W is a common mutation in the human retinal degeneration slow (RDS) gene, associated with a late-onset dominant macular dystrophy. In this study, the authors characterized a mouse model that closely mimics the human phenotype and tested the feasibility of gene supplementation as a disease treatment strategy. Transgenic mouse lines carrying the R172W mutation were generated. The retinal phenotype associated with this mutation in a low-expresser line (L-R172W) was examined, both structurally (histology with correlative immunohistochemistry) and functionally (electroretinography). By examining animals over time and with various rds genetic backgrounds, the authors evaluated the dominance of the defect. To assess the efficacy of gene transfer therapy as a treatment for this defect, a previously characterized transgenic line expressing the normal mouse peripherin/Rds (NMP) was crossed with a higher-expresser Rds line harboring the R172W mutation (H-R172W). Functional, structural, and biochemical analyses were used to assess rescue of the retinal disease phenotype. In the wild-type (WT) background, L-R172W mice exhibited late-onset (12-month) dominant cone degeneration without any apparent effect on rods. The degeneration was slightly accelerated (9 months) in the rds(+/-) background. L-R172W retinas did not form outer segments in the absence of endogenous Rds. With use of the H-R172W line on an rds(+/-) background for proof-of-principle genetic supplementation studies, the NMP transgene product rescued rod and cone functional defects and supported outer segment integrity up to 3 months of age, but the rescue effect did not persist in older (11-month) animals. The R172W mutation leads to dominant cone degeneration in the mouse model, regardless of the expression level of the transgene. In contrast, effects of the mutation on rods are dose dependent, underscoring the usefulness of the L-R172W line as a faithful model of the human phenotype. This model may prove helpful in future studies on the mechanisms of cone degeneration and for elucidating the different roles of Rds in rods and cones. This study provides evidence that Rds genetic supplementation can be used to partially rescue visual function. Although this strategy is capable of rescuing haploinsufficiency, it does not rescue the long-term degeneration associated with a gain-of-function mutation.

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

PubMed

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

2018-04-01

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

Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

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

Nifuji, Akira, E-mail: nifuji-a@tsurumi-u.ac.jp; Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama; Ideno, Hisashi

2010-04-15

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLKmore » in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.« less

Endogenous Retrovirus ev21 Dose Not Recombine with ALV-J and Induces the Expression of ISGs in the Host.

PubMed

Feng, Min; Tan, Yan; Dai, Manman; Li, Yuanfang; Xie, Tingting; Li, Hongmei; Shi, Meiqing; Zhang, Xiquan

2016-01-01

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression. Endogenous viruses integrate into host genomes and can recombine with exogenous avian leukosis virus (ALV). In this study, we analyzed the interaction of endogenous retrovirus 21 ( ev21 ) with the ALV-J in late-feathering Chinese yellow chicken. Two ALV-J strains M180 and K243 were isolated from late-feathering and fast-feathering Chinese yellow chicken flocks, respectively. The env gene of the two strains showed 94.2-94.8% nucleotide identity with reference ALV-J strains. Compared with the env gene and the LTR of ev21 and M180, the nucleotide identity of LTR was 69.7% and env gene was 58.4%, respectively, especially the amino acid identity of env gene as low as 14.2%. Phylogenetic analysis of the nucleotide sequence of the env gene and the 3'LTR showed that M180 was closely related to ALV-J, and was located in a distinct group with ev21 in the phylogenetic tree. Using co-immunoprecipitation (co-IP), we next demonstrate that the envelope protein of ev21 does not interact with the M180 envelope protein. We further show that the envelope protein of ev21 cannot activate ALV-J LTR promoter activity using luciferase-reporter assays. qPCR and western blot analysis revealed that envelope protein of endogenous ev21 can facilitate the expression of PKR at 6h post ALV-J infection (hpi) and facilitate the expression of ISG12 and CH25H at 24 hpi. However, the expression of the env gene of M180 strain was not significantly at 6 and 24 hpi. We conclude that there is no evidence of recombination between endogenous retrovirus ev21 and ALV-J strain M180 in late-feathering Chinese yellow chicken, and envelope protein of ev21 can affect the expression of host ISGs, but appears not to influence the replication of ALV-J strain M180. This is the first report of interaction among the endogenous retrovirus ev21, ALV-J and the late-feathering chicken.

KIT Signaling Promotes Growth of Colon Xenograft Tumors in Mice and is Upregulated in a Subset of Human Colon Cancers

PubMed Central

Chen, Evan C.; Karl, Taylor A.; Kalisky, Tomer; Gupta, Santosh K.; O’Brien, Catherine A.; Longacre, Teri A.; van de Rijn, Matt; Quake, Stephen R.; Clarke, Michael F.; Rothenberg, Michael E.

2015-01-01

Background & Aims Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer. Methods An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription PCR, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib following injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis. Results KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice, compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5-associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cell lines. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44+ cells indicated that KIT may promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT+ colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice. Conclusions KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors may benefit from KIT RTK inhibitors. PMID:26026391

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

PubMed

Cui, Yulin; Zhao, Jialin; Hou, Shichang; Qin, Song

2016-05-01

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

Development of B cells expressing surface immunoglobulin molecules that lack V(D)J-encoded determinants in the avian embryo bursa of Fabricius

PubMed Central

Sayegh, Camil E.; Demaries, Sandra L.; Iacampo, Sandra; Ratcliffe, Michael J. H.

1999-01-01

Immunoglobulin gene rearrangement in avian B cell precursors generates surface Ig receptors of limited diversity. It has been proposed that specificities encoded by these receptors play a critical role in B lineage development by recognizing endogenous ligands within the bursa of Fabricius. To address this issue directly we have introduced a truncated surface IgM, lacking variable region domains, into developing B precursors by retroviral gene transfer in vivo. Cells expressing this truncated receptor lack endogenous surface IgM, and the low level of endogenous Ig rearrangements that have occurred within this population of cells has not been selected for having a productive reading frame. Such cells proliferate rapidly within bursal epithelial buds of normal morphology. In addition, despite reduced levels of endogenous light chain rearrangement, those light chain rearrangements that have occurred have undergone variable region diversification by gene conversion. Therefore, although surface expression of an Ig receptor is required for bursal colonization and the induction of gene conversion, the specificity encoded by the prediversified receptor is irrelevant and, consequently, there is no obligate ligand for V(D)J-encoded determinants of prediversified avian cell surface IgM receptor. PMID:10485907

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system.

PubMed

Chang, Yizhao; Su, Tianyuan; Qi, Qingsheng; Liang, Quanfeng

2016-11-15

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid. By knocking out cas3 and activating the expression of CRISPR-associated complex for antiviral defense (Cascade), we constructed a native CRISPRi system in E. coli. Downregulation of the target gene from 6 to 82% was demonstrated using green fluorescent protein. Regulation of the citrate synthase gene (gltA) in the TCA cycle affected host metabolism. The effect of metabolic flux regulation was demonstrated by the poly-3-hydroxbutyrate (PHB) accumulation in vivo. By regulating native gltA in E. coli using an engineered endogenous type I-E CRISPR system, we redirected metabolic flux from the central metabolic pathway to the PHB synthesis pathway. This study demonstrated that the endogenous type I-E CRISPR-Cas system is an easy and effective method for regulating internal metabolic pathways, which is useful for product synthesis.

Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras.

PubMed

Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent; Sant'angelo, Derek; Sadelain, Michel

2009-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by targeting complementary sequences, referred to as miRNA recognition elements (MREs), typically located in the 3' untranslated region of mRNAs. miR-181a is highly expressed in developing thymocytes and markedly downregulated in post-thymic T cells. We investigated whether endogenous miR-181a can be harnessed to segregate expression of chimeric antigen receptors (CARs) and TCRs between developing and mature T cells. Lentiviral-encoded antigen receptors were tagged with a miR-181a-specific MRE and transduced into mouse BM cells that were used to generate hematopoietic chimeras. Expression of a CAR specific for human CD19 (hCD19) was selectively suppressed in late double-negative and double-positive thymocytes, coinciding with the peak in endogenous miR-181a expression. Receptor expression was fully restored in post-thymic resting and activated T cells, affording protection against a subsequent challenge with hCD19+ tumors. Hematopoietic mouse chimeras engrafted with a conalbumin-specific TCR prone to thymic clonal deletion acquired peptide-specific T cell responsiveness only when the vector-encoded TCR transcript was similarly engineered to be subject to regulation by miR-181a. These results demonstrate the potential of miRNA-regulated transgene expression in stem cell-based therapies, including cancer immunotherapy.

Identification of Potential Prostate Cancer-Related Pseudogenes Based on Competitive Endogenous RNA Network Hypothesis.

PubMed

Jiang, Tao; Guo, Junjie; Hu, Zhongchun; Zhao, Ming; Gu, Zhenggang; Miao, Shu

2018-06-20

BACKGROUND Long noncoding RNAs (lncRNAs) have been revealed to function as competing endogenous RNAs (ceRNAs), which can seclude the common microRNAs (miRNAs) and hence prevent the miRNAs from binding to their ancestral gene. Nonetheless, the role of lncRNA-mediated ceRNAs in prostate cancer has not yet been elucidated. MATERIAL AND METHODS Using The Cancer Genome Atlas (TCGA) database, lncRNA, miRNA, and mRNA profiles from 499 prostate cancer tissues and 52 normal prostate tissues were analyzed with the R package "DESeq" to identify the differentially expressed RNAs. GO and KEGG pathway analyses were performed using "DAVID6.8" and R packages "Clusterprofile." The ceRNA network in prostate cancer was constructed using miRDB, miRTarBase, and TargetScan databases. Survival analysis was performed with Kaplan-Meier analysis. RESULTS A total of 376 lncRNAs, 33 miRNAs, and 687 mRNAs were identified as significant factors in tumorigenesis. Based on the hypothesis that the ceRNA network (lncRNA-miRNA-mRNA regulatory axis) is involved in prostate cancer and forms competitive interrelations between miRNA and mRNA or lncRNA, we constructed a ceRNA network that included 23 lncRNAs, 6 miRNAs, and 2 mRNAs that were differentially expressed in prostate cancer. Only 3 lncRNAs (LINC00308, LINC00355, and OSTN-AS1) had a significant association with survival (P<0.05). The 3 prostate cancer-specific lncRNA were validated in prostate cancer cell lines PC3 and DU145 using qRT-PCR. CONCLUSIONS We demonstrated the differential lncRNA expression profiles in prostate cancer, which provides new insights for future studies of the ceRNA network and its regulatory mechanisms in prostate cancer.

Generation of a Slc39a8 hypomorph mouse: Markedly decreased ZIP8 Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} transporter expression

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

Wang, Bin; He, Lei; Dong, Hongbin

2011-07-01

Highlights: {yields} The mouse Slc39a8 gene encodes the ZIP8 transporter. {yields} ZIP8 functions endogenously as a electroneutral Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} symporter. {yields} A Slc39a8(neo/neo) hypomorph mouse, due to retention of the neo mini-gene, has been created. {yields} ZIP8 expression in utero is {approx}90% decreased in all tissues examined. {yields} This mouse model will be useful for studying developmental and in utero physiological functions of ZIP8. -- Abstract: Previously this laboratory has identified the mouse Slc39a8 gene encoding the ZIP8 transporter, important in cadmium uptake. ZIP8 functions endogenously as a electroneutral Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} symporter, moving bothmore » ions into the cell. The overall physiological importance of ZIP8 remains unclear. Herein we describe generation of a mouse line carrying the Slc39a8(neo) allele, containing the Frt-flanked neomycin-resistance (neo) mini-cassette in intron 3 and loxP sites in introns 3 and 6. Cre recombinase functions correctly in Escherichia coli and in adeno-Cre-infected mouse fetal fibroblasts, but does not function in the intact mouse for reasons not clear. Slc39a8(neo) is a hypomorphic allele, because Slc39a8(neo/neo) homozygotes exhibit dramatically decreased ZIP8 expression in embryo, fetus, and visceral yolk sac - in comparison to their littermate wild-type controls. This ZIP8 hypomorph will be instrumental in studying developmental and in utero physiological functions of the ZIP8 transporter.« less

CYP2S1 depletion enhances colorectal cell proliferation is associated with PGE2-mediated activation of β-catenin signaling

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

Yang, Chao; College of Life Science, Anhui Normal University, Wuhu 241000, Anhui; Li, Changyuan

2015-02-15

Colorectal epithelial cancer is one of the most common cancers in the world and its 5-year survival rate is still relatively low. Cytochrome P450 (CYP) enzymes in epithelial cells lining the alimentary tract play an important role in the oxidative metabolism of a wide range of xenobiotics, including (pro-)carcinogens and endogenous compounds. Although CYP2S1, a member of CYP family, strongly expressed in many extrahepatic tissues, the role of CYP2S1 in cancer remains unclear. To investigate whether CYP2S1 involves in colorectal carcinogenesis, cell proliferation was analyzed in HCT116 cells depleted of CYP2S1 using small hairpin interfering RNA. Our data show thatmore » CYP2S1 knockdown promotes cell proliferation through increasing the level of endogenous prostaglandin E2(PGE2). PGE2, in turn, reduces phosphorylation of β-catenin and activates β-catenin signaling, which contributes to the cell proliferation. Furthermore, CYP2S1 knockdown increase tumor growth in xenograft mouse model. In brief, these results demonstrate that CYP2S1 regulates colorectal cancer growth through associated with PGE2-mediated activation of β-catenin signaling. - Highlights: • Knockdown of CYP2S1 expression improve HCT116 cell proliferation in vitro and in vivo. • Elevate PGE2 production in CYP2S1 knockdown cell is associated with its proliferation. • Elevate PGE2 level in CYP2S1 knockdown cells enhance β-catenin accumulation. • β-catenin activate TCF/LEF and target gene expression thus promote cell proliferation.« less

Transcriptional-metabolic networks in beta-carotene-enriched potato tubers: the long and winding road to the Golden phenotype.

PubMed

Diretto, Gianfranco; Al-Babili, Salim; Tavazza, Raffaela; Scossa, Federico; Papacchioli, Velia; Migliore, Melania; Beyer, Peter; Giuliano, Giovanni

2010-10-01

Vitamin A deficiency is a public health problem in a large number of countries. Biofortification of major staple crops (wheat [Triticum aestivum], rice [Oryza sativa], maize [Zea mays], and potato [Solanum tuberosum]) with β-carotene has the potential to alleviate this nutritional problem. Previously, we engineered transgenic "Golden" potato tubers overexpressing three bacterial genes for β-carotene synthesis (CrtB, CrtI, and CrtY, encoding phytoene synthase, phytoene desaturase, and lycopene β-cyclase, respectively) and accumulating the highest amount of β-carotene in the four aforementioned crops. Here, we report the systematic quantitation of carotenoid metabolites and transcripts in 24 lines carrying six different transgene combinations under the control of the 35S and Patatin (Pat) promoters. Low levels of B-I expression are sufficient for interfering with leaf carotenogenesis, but not for β-carotene accumulation in tubers and calli, which requires high expression levels of all three genes under the control of the Pat promoter. Tubers expressing the B-I transgenes show large perturbations in the transcription of endogenous carotenoid genes, with only minor changes in carotenoid content, while the opposite phenotype (low levels of transcriptional perturbation and high carotenoid levels) is observed in Golden (Y-B-I) tubers. We used hierarchical clustering and pairwise correlation analysis, together with a new method for network correlation analysis, developed for this purpose, to assess the perturbations in transcript and metabolite levels in transgenic leaves and tubers. Through a "guilt-by-profiling" approach, we identified several endogenous genes for carotenoid biosynthesis likely to play a key regulatory role in Golden tubers, which are candidates for manipulations aimed at the further optimization of tuber carotenoid content.

Endogenous Memory CD8 T Cells Are Activated Within Cardiac Allografts Without Mediating Rejection

PubMed Central

Setoguchi, Kiyoshi; Hattori, Yusuke; Iida, Shoichi; Baldwin, William M.; Fairchild, Robert L.

2013-01-01

Endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts within 12–24 hours post-transplant in mice and are activated to proliferate and produce IFN-γ. To more accurately assess the graft injury directly imposed by these endogenous memory CD8 T cells, we took advantage of the ability of anti-LFA-1 mAb given to allograft recipients on days 3 and 4 post-transplant to inhibit the generation of primary effector T cells. When compared to grafts from IgG treated recipients on day 7 post-transplant, allografts from anti-LFA-1 mAb treated recipients had increased numbers of CD8 T cells but these grafts had marked decreases in expression levels of mRNA encoding effector mediators associated with graft injury and decreases in donor-reactive CD8 T cells producing IFN-γ. Despite this decreased activity within the allograft, CD8 T cells in allografts from recipients treated with anti-LFA-1 mAb continued to proliferate up to day 7 post-transplant and did not upregulate expression of the exhaustion marker LAG-3 but did have decreased expression of ICOS. These results indicate that endogenous memory CD8 T cells infiltrate and proliferate in cardiac allografts in mice but do not express sufficient levels of functions to mediate overt graft injury and acute rejection. PMID:23914930

The lncRNA myocardial infarction associated transcript-centric competing endogenous RNA network in non-small-cell lung cancer.

PubMed

Zheng, Chang; Li, Xuelian; Qian, Biyun; Feng, Nannan; Gao, Sumeng; Zhao, Yuxia; Zhou, Baosen

2018-01-01

The leading cause of death for cancer is lung cancer, of which the majority subtype is non-small cell lung cancer (NSCLC). Recent studies have shown long non-coding RNAs are transcribed and contribute to cancer. Previous study has shown that a few single nucleotide polymorphisms (SNPs) in myocardial infarction associated transcript (MIAT) were associated with some diseases or function as competing endogenous RNA (ceRNA) in some cancer. We performed bioinformatic methods for analyzing RNA-seq and miRNA-seq data of NSCLC from The Cancer Genome Atlas database. 1352 NSCLC patients and 1320 cancer-free controls for genotyping, and dual luciferase reporter assay, real-time PCR are performed in A549 and H1975 lung cancer cell lines. Results are analyzed by SPSS v16.0. In the present study, we focus on the role of over-expression MIAT in NSCLC. We confirmed that rs1061451 T>C (allele odds ratio = 0.22; P < 0.01) was associated with NSCLC. Furthermore, we constructed MIAT-centric ceRNA network, and three mRNAs ( MYO1B , SGK1 and WNT9A ) was identified as targets by MIAT via miR-133a-5p. C-containing genotypes of MIAT rs1061451 were protective factor of NSCLC, and MIAT, which may act as ceRNA via miR-133a-5p, modulated MYO1B , SGK1 and WNT9A expression level.

The lncRNA myocardial infarction associated transcript-centric competing endogenous RNA network in non-small-cell lung cancer

PubMed Central

Zheng, Chang; Li, Xuelian; Qian, Biyun; Feng, Nannan; Gao, Sumeng; Zhao, Yuxia; Zhou, Baosen

2018-01-01

Background The leading cause of death for cancer is lung cancer, of which the majority subtype is non-small cell lung cancer (NSCLC). Recent studies have shown long non-coding RNAs are transcribed and contribute to cancer. Previous study has shown that a few single nucleotide polymorphisms (SNPs) in myocardial infarction associated transcript (MIAT) were associated with some diseases or function as competing endogenous RNA (ceRNA) in some cancer. Patients and methods We performed bioinformatic methods for analyzing RNA-seq and miRNA-seq data of NSCLC from The Cancer Genome Atlas database. 1352 NSCLC patients and 1320 cancer-free controls for genotyping, and dual luciferase reporter assay, real-time PCR are performed in A549 and H1975 lung cancer cell lines. Results are analyzed by SPSS v16.0. Results In the present study, we focus on the role of over-expression MIAT in NSCLC. We confirmed that rs1061451 T>C (allele odds ratio = 0.22; P < 0.01) was associated with NSCLC. Furthermore, we constructed MIAT-centric ceRNA network, and three mRNAs (MYO1B, SGK1 and WNT9A) was identified as targets by MIAT via miR-133a-5p. Conclusion C-containing genotypes of MIAT rs1061451 were protective factor of NSCLC, and MIAT, which may act as ceRNA via miR-133a-5p, modulated MYO1B, SGK1 and WNT9A expression level. PMID:29795987

Endogenous Retroviruses: With Us and Against Us

NASA Astrophysics Data System (ADS)

Meyer, Thomas J.; Rosenkrantz, Jimi L.; Carbone, Lucia; Chavez, Shawn L.

2017-04-01

Mammalian genomes are scattered with thousands of copies of endogenous retroviruses (ERVs), mobile genetic elements that are relics of ancient retroviral infections. After inserting copies into the germ line of a host, most ERVs accumulate mutations that prevent the normal assembly of infectious viral particles, becoming trapped in host genomes and unable to leave to infect other cells. While most copies of ERVs are inactive, some are transcribed and encode the proteins needed to generate new insertions at novel loci. In some cases, old copies are removed via recombination and other mechanisms. This creates a shifting landscape of ERV copies within host genomes. New insertions can disrupt normal expression of nearby genes via directly inserting into key regulatory elements or by containing regulatory motifs within their sequences. Further, the transcriptional silencing of ERVs via epigenetic modification may result in changes to the epigenetic regulation of adjacent genes. In these ways, ERVs can be potent sources of regulatory disruption as well as genetic innovation. Here, we provide a brief review of the association between ERVs and gene expression, especially as observed in pre-implantation development and placentation. Moreover, we will describe the roles ERVs may play in somatic tissues, mostly in the context of human disease, including cancer, neurodegenerative disorders, and schizophrenia. Lastly, we discuss the recent discovery that some ERVs may have been pressed into the service of their host genomes to aid in the innate immune response to exogenous viral infections.

Butyrate Enhances Disease Resistance of Chickens by Inducing Antimicrobial Host Defense Peptide Gene Expression

PubMed Central

Sunkara, Lakshmi T.; Achanta, Mallika; Schreiber, Nicole B.; Bommineni, Yugendar R.; Dai, Gan; Jiang, Weiyu; Lamont, Susan; Lillehoj, Hyun S.; Beker, Ali; Teeter, Robert G.; Zhang, Guolong

2011-01-01

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. In this study, we tested the hypothesis that exogenous administration of butyrate, a major type of short-chain fatty acids derived from bacterial fermentation of undigested dietary fiber, is capable of inducing HDPs and enhancing disease resistance in chickens. We have found that butyrate is a potent inducer of several, but not all, chicken HDPs in HD11 macrophages as well as in primary monocytes, bone marrow cells, and jejuna and cecal explants. In addition, butyrate treatment enhanced the antibacterial activity of chicken monocytes against Salmonella enteritidis, with a minimum impact on inflammatory cytokine production, phagocytosis, and oxidative burst capacities of the cells. Furthermore, feed supplementation with 0.1% butyrate led to a significant increase in HDP gene expression in the intestinal tract of chickens. More importantly, such a feeding strategy resulted in a nearly 10-fold reduction in the bacterial titer in the cecum following experimental infections with S. enteritidis. Collectively, the results indicated that butyrate-induced synthesis of endogenous HDPs is a phylogenetically conserved mechanism of innate host defense shared by mammals and aves, and that dietary supplementation of butyrate has potential for further development as a convenient antibiotic-alternative strategy to enhance host innate immunity and disease resistance. PMID:22073293

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins

PubMed Central

Kennedy, Jacob J.; Abbatiello, Susan E.; Kim, Kyunggon; Yan, Ping; Whiteaker, Jeffrey R.; Lin, Chenwei; Kim, Jun Seok; Zhang, Yuzheng; Wang, Xianlong; Ivey, Richard G.; Zhao, Lei; Min, Hophil; Lee, Youngju; Yu, Myeong-Hee; Yang, Eun Gyeong; Lee, Cheolju; Wang, Pei; Rodriguez, Henry; Kim, Youngsoo; Carr, Steven A.; Paulovich, Amanda G.

2014-01-01

The successful application of MRM in biological specimens raises the exciting possibility that assays can be configured to measure all human proteins, resulting in an assay resource that would promote advances in biomedical research. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort in MRM assay generation. We have configured, validated across three laboratories, and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analyte in a panel of breast cancer-related cell lines. Median assay precision was 5.4%, with high inter-laboratory correlation (R2 >0.96). Peptide measurements in breast cancer cell lines were able to discriminate amongst molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a scaled, international effort. PMID:24317253

Adiponectin as novel regulator of cell proliferation in human glioblastoma.

PubMed

Porcile, Carola; Di Zazzo, Erika; Monaco, Maria Ludovica; D'Angelo, Giorgia; Passarella, Daniela; Russo, Claudio; Di Costanzo, Alfonso; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adriana; Zona, Gianluigi; Barbieri, Federica; Oriani, Giovannangelo; Moncharmont, Bruno; Florio, Tullio; Daniele, Aurora

2014-10-01

Adiponectin (Acrp30) is an adipocyte-secreted hormone with pleiotropic metabolic effects, whose reduced levels were related to development and progression of several malignancies. We looked at the presence of Acrp30 receptors in human glioblastomas (GBM), hypothesizing a role for Acrp30 also in this untreatable cancer. Here we demonstrate that human GBM express Acrp30 receptors (AdipoR1 and AdipoR2), which are often co-expressed in GBM samples (70% of the analyzed tumors). To investigate the effects of Acrp30 on GBM growth, we used human GBM cell lines U87-MG and U251, expressing both AdipoR1 and AdipoR2 receptors. In these cells, Acrp30 treatment inhibits DNA synthesis and cell proliferation rate, inducing arrest in G1 phase of the cell cycle. These effects were correlated to a sustained activation of ERK1/2 and Akt kinases, upon Acrp30 treatment. Our results suggest that Acrp30 may represent a novel endogenous negative regulator of GBM cell proliferation, to be evaluated for the possible development of novel pharmacological approaches. © 2014 Wiley Periodicals, Inc.

Genetic elicitation by inducible expression of β-cryptogein stimulates secretion of phenolics from Coleus blumei hairy roots.

PubMed

Vuković, Rosemary; Bauer, Nataša; Curković-Perica, Mirna

2013-02-01

The accumulation of phenolic compounds in plants is often part of the defense response against stress and pathogen attack, which can be triggered and activated by elicitors. Oomycetal proteinaceous elicitor, β-cryptogein, induces hypersensitive response and systemic acquired resistance against some pathogens. In order to test the effect of endogenously synthesized cryptogein protein on phenolic compounds accumulation in tissue, and secretion into the culture medium, Coleus blumei hairy roots were generated. Agrobacterium rhizogenes was employed to insert synthetic crypt gene, encoding β-cryptogein, under the control of alcohol-inducible promoter. The expression of β-cryptogein, in C. blumei hairy roots, was controlled by application of 1% and 2% ethanol, during 21 days induction period. Ethanol-induced expression of β-cryptogein caused significant decrease of soluble phenolics and rosmarinic acid (RA) in hairy root lines and increase of phenolics, RA and caffeic acid in culture medium. These data suggest that β-cryptogein might be a potential regulatory factor for phenolics secretion from the roots. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.