DOCSLIB.ORG

A Critical Role of Integrin-Linked Kinase, Ch-TOG and TACC3 in Centrosome Clustering in Cancer Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Critical Role of Integrin-Linked Kinase, Ch-TOG and TACC3 in Centrosome Clustering in Cancer Cells Oncogene (2011) 30, 521–534 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE A critical role of integrin-linked kinase, ch-TOG and TACC3 in centrosome clustering in cancer cells AB Fielding1, S Lim1, K Montgomery1, I Dobreva1 and S Dedhar1,2 1Department of Integrative Oncology, British Columbia Cancer Research Centre of the BC Cancer Agency, Vancouver, British Columbia, Canada and 2Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada Many cancer cells contain more than two centrosomes, Introduction which imposes a potential for multipolar mitoses, leading to cell death. To circumvent this, cancer cells develop Supernumerary centrosomes, a state in which cells mechanisms to cluster supernumerary centrosomes to contain more than two centrosomes, are a common form bipolar spindles, enabling successful mitosis. Dis- characteristic of human cancer cells. Supernumerary ruption of centrosome clustering thus provides a selective centrosomes were first observed in cancer cells nearly means of killing supernumerary centrosome-harboring 100 years ago (Boveri, 1914) and have now been cancer cells. Although the mechanisms of centrosome reported in many malignancies in vivo, including clustering are poorly understood, recent genetic analyses bladder, brain, breast, bile duct, cervical, colon, head have identified requirements for both actin and tubulin and neck, liver, lung, ovarian, pancreas and prostate regulating proteins. In this study, we demonstrate that the tumors, as well as myeloma, lymphomas and leukemias integrin-linked kinase (ILK), a protein critically involved (Lingle and Salisbury, 1999; Kramer et al., 2002; Nigg, in actin and mitotic microtubule organization, is required 2002; Pihan et al., 2003; Zyss and Gergely, 2009). for centrosome clustering. Inhibition of ILK expression or Although supernumerary centrosomes can poten- activity inhibits centrosome clustering in several breast tially facilitate tumorigenesis by disrupting cellular and prostate cancer cell lines that have centrosome polarity (Nigg, 2006; Basto et al., 2008) and increasing amplification. Furthermore, cancer cells with supernumer- the likelihood of aneuploidy (Lingle et al., 2002; Ganem ary centrosomes are significantly more sensitive to ILK et al., 2009; Silkworth et al., 2009), they also impose a inhibition than cells with two centrosomes, demonstrating potential for multipolar mitoses leading to cell death that inhibiting ILK offers a selective means of targeting (Brinkley, 2001; Nigg, 2002; Ganem et al., 2009). To cancer cells. Live cell analysis shows ILK perturbation circumvent this, many cancer cells develop mechanisms leads cancer cells to undergo multipolar anaphases, to cluster supernumerary centrosomes to form bipolar mitotic arrest and cell death in mitosis. We also show spindles and hence, ensure bipolar mitosis and cell that ILK performs its centrosome clustering activity in a survival (Ring et al., 1982; Brinkley, 2001; Quintyne focal adhesion-independent, but centrosome-dependent, et al., 2005; Rebacz et al., 2007; Kwon et al., 2008). manner through the microtubule regulating proteins Disruption of centrosome clustering and induction of TACC3 and ch-TOG. In addition, we identify a specific multipolar spindle formation thus provides a selective TACC3 phosphorylation site that is required for centro- means of killing supernumerary centrosome-harboring some clustering and demonstrate that ILK regulates this cancer cells (Kwon et al., 2008; Xu and Saunders, 2008). phosphorylation in an Aurora-A-dependent manner. Although details remain poorly understood, recent Oncogene (2011) 30, 521–534; doi:10.1038/onc.2010.431; genetic analyses have identified three broad, overlapping published online 13 September 2010 mechanisms that regulate centrosome clustering (Kwon et al., 2008). First, several microtubule-interacting/ Keywords: integrin-linked kinase; centrosome cluster- regulating genes were identified, including Ncd (human ing; multipolar mitosis; TACC3; ch-TOG/XMAP215; spleen, embryo, testes protein (HSET)) and D-TACC cancer (Kwon et al., 2008). D-TACC, a protein involved in stabilizing the minus and plus ends of microtubules at centrosomes in concert with the microtubule-associated protein ch-TOG/XMAP-215/CKAP5 (Lee et al., 2001; Barros et al., 2005), was not investigated further in this report. The second broad group of genes identified to be Correspondence: Dr S Dedhar, Department of Biochemistry, Uni- required for the prevention of multipolar mitoses were versity of British Columbia, BC Cancer Research Centre, 675 West those involved either directly in actin cytoskeleton 10th Avenue, Vancouver, British Columbia, Canada V5Z1L3. E-mail: [email protected] organization, or those which contribute to interphase Received 22 January 2010; revised 4 August 2010; accepted 9 August cell shape by linking the extra-cellular-matrix to the 2010; published online 13 September 2010 actin cytoskeleton. Centrosome clustering by ILK, ch-TOG and TACC3 AB Fielding et al 522 Integrin-linked kinase (ILK) localizes to focal adhe- markers of microtubules, centrosomes and DNA to sions and is critically involved in both actin and cell enable the analysis of multipolar spindle formation. The adhesion regulation (for reviews see Legate et al., 2006; resulting phenotypes were scored as illustrated in McDonald et al., 2008). Moreover, several reports Figure 1a and the percentage of ‘de-clustered’ centro- demonstrate that ILK has a role in mitosis by regulation somes calculated for each treatment. Approximately of the microtubule cytoskeleton. Mitotic defects have 10% of control-treated BT549s contained de-clustered been detected upon ILK depletion in Drosophila S2 cells centrosomes and multipolar spindles. This is in accor- (Bettencourt-Dias et al., 2004), mouse hepatocytes dance with data from Kwon et al, who reported 14% of (Gkretsi et al., 2007) and human glioblastoma cells control-treated BT549 cells showed multipolar spindles (Koul et al., 2005; Edwards et al., 2008). It is interesting (Kwon et al., 2008). However, ILK siRNA treatment that, although the cellular phenotypes were not reported resulted in greater than 36% of the cells displaying de- in detail in these studies, all of these systems contain clustered centrosomes (Figures 1a and c). A similar supernumerary centrosomes (Weber et al., 1998; Gui- pattern was observed in MDA-MB-231 cells and also, as dotti et al., 2003; Margall-Ducos et al., 2007; Kwon an example of a different cancer type, prostate-derived et al., 2008). ILK has also been shown to localize to PC3 cells (Figures 1b and c). These results imply that centrosomes and regulate mitotic microtubule organiza- ILK participates in the clustering of supernumerary tion, likely through its influences on Aurora-A/ch-TOG/ centrosomes in both breast and prostate cancer cells. TACC3 complex formation (Fielding et al., 2008). It These experiments were performed using pericentrin to is interesting that the ILK-interacting proteins at stain for centrosomes because of the availability of a both focal adhesions (Mig-2 (Tu et al., 2003)) and the highly specific antibody against this protein. However, centrosomes (TACC3 (Fielding et al., 2008)) have been because of the potential difficulties of distinguishing implicated in centrosome clustering (Kwon et al., 2008) between true centriole-containing centrosomes and and ILK itself has been suggested as a candidate for other sites of microtubule nucleation that may recruit regulating this process (Xu and Saunders, 2008). There- components of the soluble pericentriolar-material, such fore, we sought to test whether ILK is required for as pericentrin, multipolar spindles induced by ILK centrosome clustering and if so whether inhibiting ILK perturbation were also examined with an antibody can selectively inhibit the growth of cancer cells. against centrin 2, one of the core structural components In this study, we demonstrate that ILK is required for of centrioles. This indicated that nearly all spindle poles centrosome clustering, and inhibiting ILK expression examined in ILK-depleted cells were due to the presence with small interfering RNA (siRNA) or activity with a of true centrosomes as each stained for a pair of centrin small molecule inhibitor, induces multipolar spindles in 2 foci (Supplementary Figure 1). Quantification of the several breast and prostate cancer cell lines that have percentage of de-clustered centrosomes in ILK-depleted centrosome amplification. Furthermore, cancer cells cells as judged by centrin 2 staining gave very similar with supernumerary centrosomes are more sensitive to results to the pericentrin quantifications (Supplementary ILK inhibition than cells with two centrosomes and Figure 1c). show defects in cell division resulting in decreased cell Next, we sought to determine whether inhibiting ILK proliferation. We also found that the centrosomal kinase activity using a pharmacological ILK inhibitor proteins TACC3 and ch-TOG, which co-localize, inter- could also prevent centrosome clustering and whether act with and are regulated by ILK (Dobreva et al., 2008; this effect was specific to cancer cells. Fielding et al., 2008) are required for centrosome clustering in mammalian cells, whereas key focal adhesion binding partners of ILK are not. ILK per- Pharmacological inhibition of ILK leads to multipolar forms its centrosome-clustering functions independently
Load more

Recommended publications
  • Title a New Centrosomal Protein Regulates Neurogenesis By
    Title A new centrosomal protein regulates neurogenesis by microtubule organization Authors: Germán Camargo Ortega1-3†, Sven Falk1,2†, Pia A. Johansson1,2†, Elise Peyre4, Sanjeeb Kumar Sahu5, Loïc Broic4, Camino De Juan Romero6, Kalina Draganova1,2, Stanislav Vinopal7, Kaviya Chinnappa1‡, Anna Gavranovic1, Tugay Karakaya1, Juliane Merl-Pham8, Arie Geerlof9, Regina Feederle10,11, Wei Shao12,13, Song-Hai Shi12,13, Stefanie M. Hauck8, Frank Bradke7, Victor Borrell6, Vijay K. Tiwari§, Wieland B. Huttner14, Michaela Wilsch- Bräuninger14, Laurent Nguyen4 and Magdalena Götz1,2,11* Affiliations: 1. Institute of Stem Cell Research, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 2. Physiological Genomics, Biomedical Center, Ludwig-Maximilian University Munich, Germany. 3. Graduate School of Systemic Neurosciences, Biocenter, Ludwig-Maximilian University Munich, Germany. 4. GIGA-Neurosciences, Molecular regulation of neurogenesis, University of Liège, Belgium 5. Institute of Molecular Biology (IMB), Mainz, Germany. 6. Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández, Sant Joan d’Alacant, Spain. 7. Laboratory for Axon Growth and Regeneration, German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. 8. Research Unit Protein Science, Helmholtz Centre Munich, German Research Center for Environmental Health, Munich, Germany. 9. Protein Expression and Purification Facility, Institute of Structural Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 10. Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 11. SYNERGY, Excellence Cluster of Systems Neurology, Biomedical Center, Ludwig- Maximilian University Munich, Germany. 12. Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, USA 13.
    [Show full text]
  • Bioinformatics Identi Cation of Prognostic Factors Associated With
    Bioinformatics Identication of Prognostic Factors Associated with Breast Cancer Ying Wei Sichuan University https://orcid.org/0000-0001-8178-4705 Shipeng Zhang College of Pharmacy, North Sichuan Medical College Li Xiao West China School of Basic Medical Sciences and Forensic Medicine Jing Zou West China School of Basic Medical Sciences and Forensic Medicine Yingqing Fu West China School of Basic Medical Sciences and Forensic Medicine Yi Ye West China School of Basic Medical Sciences and Forensic Medicine Linchuan Liao ( [email protected] ) West China School of Basic Medical Sciences and Forensic Medicine https://orcid.org/0000-0003-3700-8471 Research Keywords: Breast cancer, Differentially expressed genes, miRNAs, Transcription factors, Bioinformatic analysis Posted Date: December 2nd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-117477/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/23 Abstract Background: Breast cancer (BRCA) remains one of the most common forms of cancer and is the most prominent driver of cancer-related death among women. The mechanistic basis for BRCA, however, remains incompletely understood. In particular, the relationships between driver mutations and signaling pathways in BRCA are poorly characterized, making it dicult to identify reliable clinical biomarkers that can be employed in diagnostic, therapeutic, or prognostic contexts. Methods: First, we downloaded publically available BRCA datasets (GSE45827, GSE42568, and GSE61304) from the Gene Expression Omnibus (GEO) database. We then compared gene expression proles between tumor and control tissues in these datasets using Venn diagrams and the GEO2R analytical tool. We further explore the functional relevance of BRCA-associated differentially expressed genes (DEGs) via functional and pathway enrichment analyses using the DAVID tool, and we then constructed a protein-protein interaction network incorporating DEGs of interest using the Search Tool for the Retrieval of Interacting Genes (STRING) database.
    [Show full text]
  • Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-Like Mouse Models: Tracking the Role of the Hairless Gene
    University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2006 Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene Yutao Liu University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Liu, Yutao, "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino- like Mouse Models: Tracking the Role of the Hairless Gene. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1824 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Yutao Liu entitled "Molecular and Physiological Basis for Hair Loss in Near Naked Hairless and Oak Ridge Rhino-like Mouse Models: Tracking the Role of the Hairless Gene." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Brynn H. Voy, Major Professor We have read this dissertation and recommend its acceptance: Naima Moustaid-Moussa, Yisong Wang, Rogert Hettich Accepted for the Council: Carolyn R.
    [Show full text]
  • The Basal Bodies of Chlamydomonas Reinhardtii Susan K
    Dutcher and O’Toole Cilia (2016) 5:18 DOI 10.1186/s13630-016-0039-z Cilia REVIEW Open Access The basal bodies of Chlamydomonas reinhardtii Susan K. Dutcher1* and Eileen T. O’Toole2 Abstract The unicellular green alga, Chlamydomonas reinhardtii, is a biflagellated cell that can swim or glide. C. reinhardtii cells are amenable to genetic, biochemical, proteomic, and microscopic analysis of its basal bodies. The basal bodies contain triplet microtubules and a well-ordered transition zone. Both the mother and daughter basal bodies assemble flagella. Many of the proteins found in other basal body-containing organisms are present in the Chlamydomonas genome, and mutants in these genes affect the assembly of basal bodies. Electron microscopic analysis shows that basal body duplication is site-specific and this may be important for the proper duplication and spatial organization of these organelles. Chlamydomonas is an excellent model for the study of basal bodies as well as the transition zone. Keywords: Site-specific basal body duplication, Cartwheel, Transition zone, Centrin fibers Phylogeny and conservation of proteins Centrin, SPD2/CEP192, Asterless/CEP152; CEP70, The green lineage or Viridiplantae consists of the green delta-tubulin, and epsilon-tubulin. Chlamydomonas has algae, which include Chlamydomonas, the angiosperms homologs of all of these based on sequence conservation (the land plants), and the gymnosperms (conifers, cycads, except PLK4, CEP152, and CEP192. Several lines of evi- ginkgos). They are grouped together because they have dence suggests that CEP152, CEP192, and PLK4 interact chlorophyll a and b and lack phycobiliproteins. The green [20, 52] and their concomitant absence in several organ- algae together with the cycads and ginkgos have basal isms suggests that other mechanisms exist that allow for bodies and cilia, while the angiosperms and conifers have control of duplication [4].
    [Show full text]
  • Supplemental Information Proximity Interactions Among Centrosome
    Current Biology, Volume 24 Supplemental Information Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication Elif Nur Firat-Karalar, Navin Rauniyar, John R. Yates III, and Tim Stearns Figure S1 A Myc Streptavidin -tubulin Merge Myc Streptavidin -tubulin Merge BirA*-PLK4 BirA*-CEP63 BirA*- CEP192 BirA*- CEP152 - BirA*-CCDC67 BirA* CEP152 CPAP BirA*- B C Streptavidin PCM1 Merge Myc-BirA* -CEP63 PCM1 -tubulin Merge BirA*- CEP63 DMSO - BirA* CEP63 nocodazole BirA*- CCDC67 Figure S2 A GFP – + – + GFP-CEP152 + – + – Myc-CDK5RAP2 + + + + (225 kDa) Myc-CDK5RAP2 (216 kDa) GFP-CEP152 (27 kDa) GFP Input (5%) IP: GFP B GFP-CEP152 truncation proteins Inputs (5%) IP: GFP kDa 1-7481-10441-1290218-1654749-16541045-16541-7481-10441-1290218-1654749-16541045-1654 250- Myc-CDK5RAP2 150- 150- 100- 75- GFP-CEP152 Figure S3 A B CEP63 – – + – – + GFP CCDC14 KIAA0753 Centrosome + – – + – – GFP-CCDC14 CEP152 binding binding binding targeting – + – – + – GFP-KIAA0753 GFP-KIAA0753 (140 kDa) 1-496 N M C 150- 100- GFP-CCDC14 (115 kDa) 1-424 N M – 136-496 M C – 50- CEP63 (63 kDa) 1-135 N – 37- GFP (27 kDa) 136-424 M – kDa 425-496 C – – Inputs (2%) IP: GFP C GFP-CEP63 truncation proteins D GFP-CEP63 truncation proteins Inputs (5%) IP: GFP Inputs (5%) IP: GFP kDa kDa 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl Myc- 150- Myc- 100- CCDC14 KIAA0753 100- 100- 75- 75- GFP- GFP- 50- CEP63 50- CEP63 37- 37- Figure S4 A siCtl
    [Show full text]
  • The P53/P73 - P21cip1 Tumor Suppressor Axis Guards Against Chromosomal Instability by Restraining CDK1 in Human Cancer Cells
    Oncogene (2021) 40:436–451 https://doi.org/10.1038/s41388-020-01524-4 ARTICLE The p53/p73 - p21CIP1 tumor suppressor axis guards against chromosomal instability by restraining CDK1 in human cancer cells 1 1 2 1 2 Ann-Kathrin Schmidt ● Karoline Pudelko ● Jan-Eric Boekenkamp ● Katharina Berger ● Maik Kschischo ● Holger Bastians 1 Received: 2 July 2020 / Revised: 2 October 2020 / Accepted: 13 October 2020 / Published online: 9 November 2020 © The Author(s) 2020. This article is published with open access Abstract Whole chromosome instability (W-CIN) is a hallmark of human cancer and contributes to the evolvement of aneuploidy. W-CIN can be induced by abnormally increased microtubule plus end assembly rates during mitosis leading to the generation of lagging chromosomes during anaphase as a major form of mitotic errors in human cancer cells. Here, we show that loss of the tumor suppressor genes TP53 and TP73 can trigger increased mitotic microtubule assembly rates, lagging chromosomes, and W-CIN. CDKN1A, encoding for the CDK inhibitor p21CIP1, represents a critical target gene of p53/p73. Loss of p21CIP1 unleashes CDK1 activity which causes W-CIN in otherwise chromosomally stable cancer cells. fi Vice versa 1234567890();,: 1234567890();,: Consequently, induction of CDK1 is suf cient to induce abnormal microtubule assembly rates and W-CIN. , partial inhibition of CDK1 activity in chromosomally unstable cancer cells corrects abnormal microtubule behavior and suppresses W-CIN. Thus, our study shows that the p53/p73 - p21CIP1 tumor suppressor axis, whose loss is associated with W-CIN in human cancer, safeguards against chromosome missegregation and aneuploidy by preventing abnormally increased CDK1 activity.
    [Show full text]
  • Cep57, a NEDD1-Binding Pericentriolar Material Component, Is Essential for Spindle Pole Integrity
    Cell Research (2012) :1-12. © 2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr Cep57, a NEDD1-binding pericentriolar material component, is essential for spindle pole integrity Qixi Wu1, *, Runsheng He1, *, Haining Zhou1, Albert CH Yu2, Bo Zhang1, Junlin Teng1, Jianguo Chen1, 3 1The State Key Laboratory of Biomembrane and Membrane Bioengineering and The Key Laboratory of Cell Proliferation and Dif- ferentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China; 2Department of Neurobi- ology, Neuroscience Research Institute, School of Basic Medical Sciences, Peking University, Beijing 100191, China; 3The Center for Theoretical Biology, Peking University, Beijing 100871, China Formation of a bipolar spindle is indispensable for faithful chromosome segregation and cell division. Spindle in- tegrity is largely dependent on the centrosome and the microtubule network. Centrosome protein Cep57 can bundle microtubules in mammalian cells. Its related protein (Cep57R) in Xenopus was characterized as a stabilization factor for microtubule-kinetochore attachment. Here we show that Cep57 is a pericentriolar material (PCM) component. Its interaction with NEDD1 is necessary for the centrosome localization of Cep57. Depletion of Cep57 leads to unaligned chromosomes and a multipolar spindle, which is induced by PCM fragmentation. In the absence of Cep57, cen- trosome microtubule array assembly activity is weakened, and the spindle length and microtubule density decrease. As a spindle microtubule-binding protein, Cep57 is also responsible for the proper organization of the spindle micro- tubule and localization of spindle pole focusing proteins. Collectively, these results suggest that Cep57, as a NEDD1- binding centrosome component, could function as a spindle pole- and microtubule-stabilizing factor for establishing robust spindle architecture.
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • Supplementary Data
    SUPPLEMENTARY DATA A cyclin D1-dependent transcriptional program predicts clinical outcome in mantle cell lymphoma Santiago Demajo et al. 1 SUPPLEMENTARY DATA INDEX Supplementary Methods p. 3 Supplementary References p. 8 Supplementary Tables (S1 to S5) p. 9 Supplementary Figures (S1 to S15) p. 17 2 SUPPLEMENTARY METHODS Western blot, immunoprecipitation, and qRT-PCR Western blot (WB) analysis was performed as previously described (1), using cyclin D1 (Santa Cruz Biotechnology, sc-753, RRID:AB_2070433) and tubulin (Sigma-Aldrich, T5168, RRID:AB_477579) antibodies. Co-immunoprecipitation assays were performed as described before (2), using cyclin D1 antibody (Santa Cruz Biotechnology, sc-8396, RRID:AB_627344) or control IgG (Santa Cruz Biotechnology, sc-2025, RRID:AB_737182) followed by protein G- magnetic beads (Invitrogen) incubation and elution with Glycine 100mM pH=2.5. Co-IP experiments were performed within five weeks after cell thawing. Cyclin D1 (Santa Cruz Biotechnology, sc-753), E2F4 (Bethyl, A302-134A, RRID:AB_1720353), FOXM1 (Santa Cruz Biotechnology, sc-502, RRID:AB_631523), and CBP (Santa Cruz Biotechnology, sc-7300, RRID:AB_626817) antibodies were used for WB detection. In figure 1A and supplementary figure S2A, the same blot was probed with cyclin D1 and tubulin antibodies by cutting the membrane. In figure 2H, cyclin D1 and CBP blots correspond to the same membrane while E2F4 and FOXM1 blots correspond to an independent membrane. Image acquisition was performed with ImageQuant LAS 4000 mini (GE Healthcare). Image processing and quantification were performed with Multi Gauge software (Fujifilm). For qRT-PCR analysis, cDNA was generated from 1 µg RNA with qScript cDNA Synthesis kit (Quantabio). qRT–PCR reaction was performed using SYBR green (Roche).
    [Show full text]
  • AURKA, DLGAP5, TPX2, KIF11 and CKAP5: Five Specific Mitosis-Associated Genes Correlate with Poor Prognosis for Non-Small Cell Lung Cancer Patients
    INTERNATIONAL JOURNAL OF ONCOLOGY 50: 365-372, 2017 AURKA, DLGAP5, TPX2, KIF11 and CKAP5: Five specific mitosis-associated genes correlate with poor prognosis for non-small cell lung cancer patients MARC A. SCHNEIDER1,8, PETROS CHristopoulos2,8, THOMAS MULEY1,8, ARNE WartH5,8, URSULA KLINGMUELLER6,8,9, MICHAEL THOMAS2,8, FELIX J.F. HertH3,8, HENDRIK DIENEMANN4,8, NIKOLA S. MUELLER7,9, FABIAN THEIS7,9 and MICHAEL MEISTER1,8,9 1Translational Research Unit, 2Department of Thoracic Oncology, 3Department of Pneumology and Critical Care Medicine, and 4Department of Surgery, Thoraxklinik at University Hospital Heidelberg, Heidelberg; 5Institute of Pathology, University of Heidelberg, Heidelberg; 6Systems Biology of Signal Transduction, German Cancer Research Center, Heidelberg; 7Cellular Dynamics and Cell Patterning, Max Planck Institute of Biochemistry, Martinsried; 8Translational Lung Research Center Heidelberg (TLRC-H), Member of the German Center for Lung Research (DZL) Heidelberg; 9CancerSys network ‘LungSysII’, Heidelberg, Germany Received October 26, 2016; Accepted December 5, 2016 DOI: 10.3892/ijo.2017.3834 Abstract. The growth of a tumor depends to a certain extent genes AURKA, DLGAP5, TPX2, KIF11 and CKAP5 is asso- on an increase in mitotic events. Key steps during mitosis are ciated with the prognosis of NSCLC patients. the regulated assembly of the spindle apparatus and the sepa- ration of the sister chromatids. The microtubule-associated Introduction protein Aurora kinase A phosphorylates DLGAP5 in order to correctly segregate the chromatids. Its activity and recruitment Lung cancer is globally the leading cause of cancer-related to the spindle apparatus is regulated by TPX2. KIF11 and deaths (1). Non-small cell lung cancer (NSCLC), which accounts CKAP5 control the correct arrangement of the microtubules for more than 80% of all cases, is divided in adenocarcinoma and prevent their degradation.
    [Show full text]
  • Downloaded the “Top Edge” Version
    bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Drosophila models of pathogenic copy-number variant genes show global and 2 non-neuronal defects during development 3 Short title: Non-neuronal defects of fly homologs of CNV genes 4 Tanzeen Yusuff1,4, Matthew Jensen1,4, Sneha Yennawar1,4, Lucilla Pizzo1, Siddharth 5 Karthikeyan1, Dagny J. Gould1, Avik Sarker1, Yurika Matsui1,2, Janani Iyer1, Zhi-Chun Lai1,2, 6 and Santhosh Girirajan1,3* 7 8 1. Department of Biochemistry and Molecular Biology, Pennsylvania State University, 9 University Park, PA 16802 10 2. Department of Biology, Pennsylvania State University, University Park, PA 16802 11 3. Department of Anthropology, Pennsylvania State University, University Park, PA 16802 12 4 contributed equally to work 13 14 *Correspondence: 15 Santhosh Girirajan, MBBS, PhD 16 205A Life Sciences Building 17 Pennsylvania State University 18 University Park, PA 16802 19 E-mail: [email protected] 20 Phone: 814-865-0674 21 1 bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 22 ABSTRACT 23 While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non- 24 neuronal phenotypes, functional studies evaluating these regions have focused on the molecular 25 basis of neuronal defects.
    [Show full text]
  • Downloaded from Bioscientifica.Com at 10/04/2021 03:09:50PM Via Free Access
    242 S-M Ho et al. Regulation of centrosome 24:2 83–96 Research duplication by BPA analogues Bisphenol A and its analogues disrupt centrosome cycle and microtubule dynamics in prostate cancer Shuk-Mei Ho1,2,3,4, Rahul Rao1, Sarah To1,5,6, Emma Schoch1 and Pheruza Tarapore1,2,3 1Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio, USA 2Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, Ohio, USA Correspondence 3Cincinnati Cancer Center, Cincinnati, Ohio, USA should be addressed 4Cincinnati Veteran Affairs Hospital Medical Center, Cincinnati, Ohio, USA to S-M Ho or P Tarapore 5Center for Cancer Research, Hudson Institute of Medical Research, Clayton, Victoria, Australia Email 6Monash University, Clayton, Victoria, Australia [email protected] or [email protected] Abstract Humans are increasingly exposed to structural analogues of bisphenol A (BPA), as Key Words BPA is being replaced by these compounds in BPA-free consumer products. We have f endocrine-disrupting previously shown that chronic and developmental exposure to BPA is associated with chemicals increased prostate cancer (PCa) risk in human and animal models. Here, we examine f bisphenol A analogues whether exposure of PCa cells (LNCaP, C4-2) to low-dose BPA and its structural analogues f BPA (BPS, BPF, BPAF, TBBPA, DMBPA and TMBPA) affects centrosome amplification (CA), f BPS a hallmark of cancer initiation and progression. We found that exposure to BPA, BPS, f BPF DMBPA and TBBPA, in descending order, increased the number of cells with CA, in a non- f TBBPA Endocrine-Related Cancer Endocrine-Related monotonic dose–response manner.
    [Show full text]