Browsing by Subject "HUMAN-CELLS"

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  • Arczewska, Katarzyna D.; Tomazella, Gisele G.; Lindvall, Jessica M.; Kassahun, Henok; Maglioni, Silvia; Torgovnick, Alessandro; Henriksson, Johan; Matilainen, Olli; Marquis, Bryce J.; Nelson, Bryant C.; Jaruga, Pawel; Babaie, Eshrat; Holmberg, Carina; Burglin, Thomas R.; Ventura, Natascia; Thiede, Bernd; Nilsen, Hilde (2013)
  • El-Khoury, Riyad; Dufour, Eric; Rak, Malgorzata; Ramanantsoa, Nelina; Grandchamp, Nicolas; Csaba, Zsolt; Duvillie, Bertrand; Benit, Paule; Gallego, Jorge; Gressens, Pierre; Sarkis, Chamsy; Jacobs, Howard T.; Rustin, Pierre (2013)
  • Hamdi, Yosr; Soucy, Penny; Kuchenbaeker, Karoline B.; Pastinen, Tomi; Droit, Arnaud; Lemacon, Audrey; Adlard, Julian; Aittomäki, Kristiina; Andrulis, Irene L.; Arason, Adalgeir; Arnold, Norbert; Arun, Banu K.; Azzollini, Jacopo; Bane, Anita; Barjhoux, Laure; Barrowdale, Daniel; Benitez, Javier; Berthet, Pascaline; Blok, Marinus J.; Bobolis, Kristie; Bonadona, Valerie; Bonanni, Bernardo; Bradbury, Angela R.; Brewer, Carole; Buecher, Bruno; Buys, Saundra S.; Caligo, Maria A.; Chiquette, Jocelyne; Chung, Wendy K.; Claes, Kathleen B. M.; Daly, Mary B.; Damiola, Francesca; Davidson, Rosemarie; De la Hoya, Miguel; De Leeneer, Kim; Diez, Orland; Ding, Yuan Chun; Dolcetti, Riccardo; Domchek, Susan M.; Dorfling, Cecilia M.; Eccles, Diana; Eeles, Ros; Einbeigi, Zakaria; Ejlertsen, Bent; Engel, Christoph; Evans, D. Gareth; Feliubadalo, Lidia; Foretova, Lenka; Fostira, Florentia; Nevanlinna, Heli; EMBRACE; GEMO Study Collaborators; HEBON; kConFab Investigators (2017)
    Cis-acting regulatory SNPs resulting in differential allelic expression (DAE) may, in part, explain the underlying phenotypic variation associated with many complex diseases. To investigate whether common variants associated with DAE were involved in breast cancer susceptibility among BRCA1 and BRCA2 mutation carriers, a list of 175 genes was developed based of their involvement in cancer-related pathways. Using data from a genome-wide map of SNPs associated with allelic expression, we assessed the association of similar to 320 SNPs located in the vicinity of these genes with breast and ovarian cancer risks in 15,252 BRCA1 and 8211 BRCA2 mutation carriers ascertained from 54 studies participating in the Consortium of Investigators of Modifiers of BRCA1/2. We identified a region on 11q22.3 that is significantly associated with breast cancer risk in BRCA1 mutation carriers (most significant SNP rs228595 p = 7 x 10(-6)). This association was absent in BRCA2 carriers (p = 0.57). The 11q22.3 region notably encompasses genes such as ACAT1, NPAT, and ATM. Expression quantitative trait loci associations were observed in both normal breast and tumors across this region, namely for ACAT1, ATM, and other genes. In silico analysis revealed some overlap between top risk-associated SNPs and relevant biological features in mammary cell data, which suggests potential functional significance. We identified 11q22.3 as a new modifier locus in BRCA1 carriers. Replication in larger studies using estrogen receptor (ER)-negative or triple-negative (i.e., ER-, progesterone receptor-, and HER2-negative) cases could therefore be helpful to confirm the association of this locus with breast cancer risk.
  • Szibor, Marten; Dhandapani, Praveen K.; Dufour, Eric; Holmstrom, Kira M.; Zhuang, Yuan; Salwig, Isabelle; Wittig, Ilka; Heidler, Juliana; Gizatullina, Zemfira; Gainutdinov, Timur; Fuchs, Helmut; Gailus-Durner, Valerie; de Angelis, Martin Hrabe; Nandania, Jatin; Velagapudi, Vidya; Wietelmann, Astrid; Rustin, Pierre; Gellerich, Frank N.; Jacobs, Howard T.; Braun, Thomas; German Mouse Clinic Consortium (2017)
    Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOX(Rosa26) mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOX(Rosa26) mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.
  • Gospodaryov, Dmytro V.; Lushchak, Oleh V.; Rovenko, Bohdana M.; Perkhulyn, Natalia V.; Gerards, Mike; Tuomela, Tea; Jacobs, Howard T. (2014)
  • Schmierer, Bernhard; Botla, Sandeep K.; Zhang, Jilin; Turunen, Mikko; Kivioja, Teemu; Taipale, Jussi (2017)
    Loss-of-function screening by CRISPR/Cas9 gene knockout with pooled, lentiviral guide libraries is a widely applicable method for systematic identification of genes contributing to diverse cellular phenotypes. Here, Random Sequence Labels (RSLs) are incorporated into the guide library, which act as unique molecular identifiers (UMIs) to allow massively parallel lineage tracing and lineage dropout screening. RSLs greatly improve the reproducibility of results by increasing both the precision and the accuracy of screens. They reduce the number of cells needed to reach a set statistical power, or allow a more robust screen using the same number of cells.
  • Pohjoismaki, Jaakko L. O.; Goffart, Steffi; Taylor, Robert W.; Turnbull, Douglas M.; Suomalainen, Anu; Jacobs, Howard T.; Karhunen, Pekka J. (2010)
  • Rodrigues, Ana Paula C.; Camargo, Andre F.; Andjelkovic, Ana; Jacobs, Howard T.; Oliveira, Marcos T. (2018)
    The xenotopic expression of the alternative oxidase AOX from the tunicate Ciona intestinalis in diverse models of human disease partially alleviates the phenotypic effects of mitochondrial respiratory chain defects. AOX is a non-proton pumping, mitochondrial inner membrane-bound, single-subunit enzyme that can bypass electron transport through the cytochrome segment, providing an additional site for ubiquinone reoxidation and oxygen reduction upon respiratory chain overload. We set out to investigate whether AOX expression in Drosophila could counteract the effects of mitochondrial DNA (mtDNA) replication defects caused by disturbances in the mtDNA helicase or DNA polymerase gamma. We observed that the developmental arrest imposed by either the expression of mutant forms of these enzymes or their knockdown was not rescued by AOX. Considering also the inability of AOX to ameliorate the phenotype of tko(25t), a fly mutant with mitochondrial translation deficiency, we infer that this alternative enzyme may not be applicable to cases of mitochondrial gene expression defects. Finding the limitations of AOX applicability will help establish the parameters for the future putative use of this enzyme in gene therapies for human mitochondrial diseases.
  • Andjelkovic, Ana; Oliveira, Marcos T.; Cannino, Giuseppe; Yalgin, Cagri; Dhandapani, Praveen K.; Dufour, Eric; Rustin, Pierre; Szibor, Marten; Jacobs, Howard T. (2015)
    The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.
  • Saari, Sina; Andjelkovic, Ana; Garcia, Geovana S.; Jacobs, Howard T.; Oliveira, Marcos T. (2017)
    Background: Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question. Methods: Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the a-tubulin promoter, and wild-type males of the same genetic background. Results: In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation. Conclusions: AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.
  • Wucher, Valentin; Legeai, Fabrice; Hedan, Benoit; Rizk, Guillaume; Lagoutte, Laetitia; Leeb, Tosso; Jagannathan, Vidhya; Cadieu, Edouard; David, Audrey; Lohi, Hannes; Cirera, Susanna; Fredholm, Merete; Botherel, Nadine; Leegwater, Peter A. J.; Le Beguec, Celine; Fieten, Hille; Johnson, Jeremy; Alfoldi, Jessica; Andre, Catherine; Lindblad-Toh, Kerstin; Hitte, Christophe; Derrien, Thomas (2017)
    Whole transcriptome sequencing (RNA-seq) has become a standard for cataloguing and monitoring RNA populations. One of the main bottlenecks, however, is to correctly identify the different classes of RNAs among the plethora of reconstructed transcripts, particularly those that will be translated (mRNAs) from the class of long non-coding RNAs (lncRNAs). Here, we present FEELnc (FlExible Extraction of LncRNAs), an alignment-free program that accurately annotates lncRNAs based on a Random Forest model trained with general features such as multi k-mer frequencies and relaxed open reading frames. Benchmarking versus five state-of-the-art tools shows that FEELnc achieves similar or better classification performance on GENCODE and NONCODE data sets. The program also provides specific modules that enable the user to fine-tune classification accuracy, to formalize the annotation of lncRNA classes and to identify lncRNAs even in the absence of a training set of non-coding RNAs. We used FEELnc on a real data set comprising 20 canine RNA-seq samples produced by the European LUPA consortium to substantially expand the canine genome annotation to include 10 374 novel lncRNAs and 58 640 mRNA transcripts. FEELnc moves beyond conventional coding potential classifiers by providing a standardized and complete solution for annotating lncRNAs and is freely available at https://github.com/tderrien/FEELnc.
  • Lippert, Timothy P.; Marzec, Paulina; Idilli, Aurora I.; Sarek, Grzegorz; Vancevska, Aleksandra; Bower, Mark; Farrell, Paul J.; Ojala, Päivi M.; Feldhahn, Niklas; Boulton, Simon J. (2021)
    To achieve replicative immortality, cancer cells must activate telomere maintenance mechanisms to prevent telomere shortening. similar to 85% of cancers circumvent telomeric attrition by re-expressing telomerase, while the remaining similar to 15% of cancers induce alternative lengthening of telomeres (ALT), which relies on break-induced replication (BIR) and telomere recombination. Although ALT tumours were first reported over 20 years ago, the mechanism of ALT induction remains unclear and no study to date has described a cell-based model that permits the induction of ALT. Here, we demonstrate that infection with Kaposi's sarcoma herpesvirus (KSHV) induces sustained acquisition of ALT-like features in previously non-ALT cell lines. KSHV-infected cells acquire hallmarks of ALT activity that are also observed in KSHV-associated tumour biopsies. Down-regulating BIR impairs KSHV latency, suggesting that KSHV co-opts ALT for viral functionality. This study uncovers KSHV infection as a means to study telomere maintenance by ALT and reveals features of ALT in KSHV-associated tumours. similar to 15% of cancers induce alternative lengthening of telomeres (ALT) to activate telomere maintenance. Here, the authors reveal that infection with Kaposi's sarcoma herpesvirus (KSHV) induces acquisition of ALT-like features in previously non-ALT cell lines.
  • Ciesielski, Grzegorz L.; Nadalutti, Cristina A.; Oliveira, Marcos T.; Jacobs, Howard T.; Griffith, Jack D.; Kaguni, Laurie S. (2018)
    Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of themitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization.
  • Zimmer, Jutta; Tacconi, Eliana M. C.; Folio, Cecilia; Badie, Sophie; Porru, Manuela; Klare, Kerstin; Tumiati, Manuela; Markkanen, Enni; Halder, Swagata; Ryan, Anderson; Jackson, Stephen P.; Ramadan, Kristijan; Kuznetsov, Sergey G.; Biroccio, Annamaria; Sale, Julian E.; Tarsounas, Madalena (2016)
    G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.
  • Balistreri, Giuseppe; Bognanni, Claudia; Mhlemann, Oliver (2017)
    Nonsense-mediated mRNA decay (NMD), a cellular RNA turnover pathway targeting RNAs with features resulting in aberrant translation termination, has recently been found to restrict the replication of positive-stranded RNA((+) RNA) viruses. As for every other antiviral immune system, there is also evidence of viruses interfering with and modulating NMD to their own advantage. This review will discuss our current understanding of why and how NMD targets viral RNAs, and elaborate counter-defense strategies viruses utilize to escape NMD.