DOCSLIB.ORG

Identification of Necrosis-Associated Genes in Glioblastoma by Cdna Microarray Analysis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Identification of Necrosis-Associated Genes in Glioblastoma by Cdna Microarray Analysis 212 Vol. 10, 212–221, January 1, 2004 Clinical Cancer Research Identification of Necrosis-Associated Genes in Glioblastoma by cDNA Microarray Analysis Shaan M. Raza,1 Gregory N. Fuller,2 the 26 genes between grade III necrosis GBM and anaplastic Chang Hun Rhee,4 Suyun Huang,1 astrocytoma (AA) samples; all but 1 of the genes had ele- Kenneth Hess,3 Wei Zhang,2 and vated expression when comparing necrosis grade III with 1 AA samples. Two factors, the ephrin type A receptor 1 and Raymond Sawaya the prostaglandin E receptor EP4 subtype, not previously 1 2 3 2 Departments of Neurosurgery, Pathology, and Biostatistics, Brain considered in this context, were highlighted because of their Tumor Center, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, and 4Department of Neurosurgery, Korean particularly high (positive) correlation coefficients; immu- Cancer Center Hospital, Seoul, Korea nostaining showed the products of these two genes to be localized in perinecrotic and necrotic regions and to be overexpressed in grade III GBMs, but not AAs. These two ABSTRACT molecules also showed significant correlation with survival .in a combined model (0.0034 ؍ Purpose: In the field of cancer research, there has been of GBM patients (P a paucity of interest in necrosis, whereas studies focusing on Conclusions: The application of cDNA expression mi- apoptosis abound. In neuro-oncology, this is particularly croarray analysis has identified specific genes and patterns surprising because of the importance of necrosis as a hall- of gene expression that may help elucidate the molecular mark of glioblastoma (GBM), the most malignant and most basis of necrogenesis in GBM. Additional studies will be common primary brain tumor, and the fact that the degree required to further investigate and confirm these findings. of necrosis has been shown to be inversely related to patient survival. It is therefore of considerable interest and impor- tance to identify genes and gene products related to necrosis INTRODUCTION formation. In cancer research, the subject of cell death has been Experimental Design: We used a nylon cDNA microar- extensively studied with the goal of achieving therapeutically ray to analyze mRNA expression of 588 universal cellular induced tumor death. Among the two major forms of cell death genes in 15 surgically resected human GBM samples with encountered in biology, apoptosis has received a proportionately varying degrees of necrosis. Gene expression was correlated greater degree of attention and emphasis than has necrosis (1, 2). with the degree of necrosis using rank correlation coeffi- A large body of recent literature has provided an understanding cients. The expression of identified genes was compared with of the physiological and molecular events that lead to apoptosis; their expression in tissue samples from 5 anaplastic astro- yet similar characterization of necrosis cannot be found. cytomas (AAs). Immunostaining was used to determine In the field of neuro-oncology, this paucity of interest in whether genes showing the most positive correlation with necrosis is surprising because it is a hallmark of the most necrosis were increasingly expressed in tumor tissues, as common and most malignant primary brain tumor—glioblas- grade of necrosis increased. toma (GBM). In fact, GBM is differentially diagnosed from Results: The hybridization results indicated that 26 lower grade astrocytomas based on the histological presence of genes showed significant correlation with the amount of de novo tumor necrosis and associated microvascular prolifer- necrosis. All 26 genes had functions associated with either ation. Clinical studies indicate that of all clinical, neuroimaging, Ras, Akt, tumor necrosis factor ␣, nuclear factor ␬B, apo- and histopathological characteristics (including age), necrosis ptosis, procoagulation, or hypoxia. Nine genes were posi- that is visible on magnetic resonance imaging scans has the tively correlated with necrosis grade, and 17 genes were greatest prognostic value and is inversely related to patient negatively correlated with necrosis grade. There were sig- survival (3). Based on the clinical implications and potential for nificant differences in the median expression levels of 3 of novel therapeutic interventions, the molecular factors mediating necrogenesis in GBM warrant investigation. An initial step in this process would be the identification of specific gene prod- ucts associated with necrosis. Received 1/28/03; revised 9/11/03; accepted 9/16/03. Although there have been a few limited studies of one or Grant support: Texas Higher Education Coordinating Board (to W. Z. two necrosis-associated molecules, there have been no efforts to and G. N. F.) and the Anthony Bullock III Brain Tumor Research Fund characterize global gene expression changes associated with (to R. S.). The costs of publication of this article were defrayed in part by the varying degrees of necrosis in GBM (4). In this study, we used payment of page charges. This article must therefore be hereby marked cDNA expression microarrays to identify the molecular finger- advertisement in accordance with 18 U.S.C. Section 1734 solely to prints of 15 GBM samples with differing degrees of necrosis; indicate this fact. this was done with the goal of identifying genes whose mRNA Requests for reprints: Raymond Sawaya, Department of Neurosur- gery–442, The University of Texas M. D. Anderson Cancer Center, expression is correlated with the formation of necrosis. In hopes 1515 Holcombe Boulevard, Houston, Texas 77030. Phone: (713) 792- of establishing a relationship between necrosis and the identified 2400; Fax: (713) 794-4950; E-mail: [email protected]. genes, their expression levels in anaplastic astrocytomas (AAs), Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 213 which by definition (St. Anne/Mayo criteria) are tumors that are Table 1 Necrosis grades of human glioblastoma samplesa and not associated with necrosis, were determined and compared survival time of patients with expression levels in grade III necrosis GBM samples, Necrosis Sample Median survival which are tumors characterized by extensive amounts of necrosis. grade no. (weeks) 04 64 MATERIALS AND METHODS I 2 116 II 5 53 Primary Glioma Tissues. All human tumor samples III 4 25 were obtained from the Brain Tumor Center Tissue Bank at The a The following grading system (see Ref. 3) was used: grade 0, no University of Texas M. D. Anderson Cancer Center (Houston, necrosis apparent on the magnetic resonance imaging scan; grade I, TX). After surgical removal, tissue samples were quickly frozen amount of necrosis is less than 25% of the tumor volume; grade II, at Ϫ80°C. All tissue samples used for cDNA analysis were amount of necrosis is between 25% and 50% of the tumor volume; grade evaluated by a neuropathologist; all specimens selected were III, amount of necrosis is greater than 50% of the tumor volume. In all characterized by dense tumor cellularity and the presence of cases, the extent of necrosis was confirmed at surgery. Ͻ10% normal brain tissue. A second neuropathologist con- firmed all diagnoses. Grading of astrocytic neoplasms was per- formed according to the St. Anne/Mayo criteria. A total of 5 AAs and 15 GBM samples were analyzed. The 15 GBM sam- volume (Ref. 3; Fig. 1). The extent of necrosis was confirmed in ples obtained were further graded based on the amount of all cases during surgery. Within our sample population, four necrosis observed on magnetic resonance imaging scans. Ne- tumors were grade 0, two were grade I, five were grade II, crosis on magnetic resonance imaging scans appears as a and four were grade III. The sample distribution is shown in hypointense region of T1 signal surrounded by a contrast- Table 1. enhanced region representing viable tumor. Necrosis was graded Isolation of Total RNA and mRNA from Tissue Sam- according to the following previously described system: grade 0, ples. The frozen tissue samples were ground to a powder in no necrosis apparent on the magnetic resonance imaging scan; liquid nitrogen using a mortar and pestle and then treated with grade I, amount of necrosis is Ͻ25% of the tumor volume; grade lysis buffer, TRI Reagent (Molecular Research Center, Cincin- II, amount of necrosis is between 25% and 50% of the tumor nati, OH), to extract nucleic acids and prevent their degradation. volume; grade III, amount of necrosis is Ͼ50% of the tumor Total RNA was isolated as described previously (5). The quality of this RNA was checked by electrophoresis on a denaturing formaldehyde agarose gel. The presence of high-quality unde- graded RNA was verified by the observation of a lack of streaking on the lower (anodal) part of the sample lane on the gel. A further indication that high-quality RNA was obtained was the presence on the gel of a 28S rRNA band twice as intense as that of the 18S rRNA band. A poly(dT) mRNA isolation column (Qiagen, Inc., Chatsworth, CA) was used to separate mRNA from high-quality total RNA. With this column, we recovered 1–2 ␮g of mRNA from approximately 200 ␮g of total RNA, but only 0.5–1.0 ␮g of mRNA was required for analysis in the cDNA array. In this study, we obtained high-quality RNA from Ͼ90% of the resected samples. Because undegraded RNA is scarce within regions of necrosis, total RNA and mRNA were predominantly isolated from viable nonnecrotic tumor regions. Hybridization to Human Atlas cDNA Expression Array Blots. cDNA fragments, approximately 200- to 500-bp long, representing 588 human genes with known functions and tight transcriptional controls were immobilized in duplicate on a nylon membrane (Clontech Laboratories, Inc.). To minimize cross-hybridization and nonspecific binding of cDNA probes, each fragment was selected as a unique sequence without a poly(A) tail, highly homologous sequences, or repetitive ele- ments. 32P-labeled cDNA probes were generated through re- verse transcription of 0.5–1.0 ␮g of each analyzed poly(A)ϩ Fig.
Load more

Recommended publications
  • To Study Mutant P53 Gain of Function, Various Tumor-Derived P53 Mutants
    Differential effects of mutant TAp63γ on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science By Shama K Khokhar M.Sc., Bilaspur University, 2004 B.Sc., Bhopal University, 2002 2007 1 COPYRIGHT SHAMA K KHOKHAR 2007 2 WRIGHT STATE UNIVERSITY SCHOOL OF GRADUATE STUDIES Date of Defense: 12-03-07 I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY SHAMA KHAN KHOKHAR ENTITLED Differential effects of mutant TAp63γ on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science Madhavi P. Kadakia, Ph.D. Thesis Director Daniel Organisciak , Ph.D. Department Chair Committee on Final Examination Madhavi P. Kadakia, Ph.D. Steven J. Berberich, Ph.D. Michael Leffak, Ph.D. Joseph F. Thomas, Jr., Ph.D. Dean, School of Graduate Studies 3 Abstract Khokhar, Shama K. M.S., Department of Biochemistry and Molecular Biology, Wright State University, 2007 Differential effect of TAp63γ mutants on transactivation of p53 and/or p63 responsive genes and their effects on global gene expression. p63, a member of the p53 gene family, known to play a role in development, has more recently also been implicated in cancer progression. Mice lacking p63 exhibit severe developmental defects such as limb truncations, abnormal skin, and absence of hair follicles, teeth, and mammary glands. Germline missense mutations of p63 have been shown to be responsible for several human developmental syndromes including SHFM, EEC and ADULT syndromes and are associated with anomalies in the development of organs of epithelial origin.
    [Show full text]
  • HOXB6 Homeo Box B6 HOXB5 Homeo Box B5 WNT5A Wingless-Type
    5 6 6 5 . 4 2 1 1 1 2 4 6 4 3 2 9 9 7 0 5 7 5 8 6 4 0 8 2 3 1 8 3 7 1 0 0 4 0 2 5 0 8 7 5 4 1 1 0 3 6 0 4 8 3 7 4 7 6 9 6 7 1 5 0 8 1 4 1 1 7 1 0 0 4 2 0 8 1 1 1 2 5 3 5 0 7 2 6 9 1 2 1 8 3 5 2 9 8 0 6 0 9 5 1 9 9 2 1 1 6 0 2 3 0 3 6 9 1 6 5 5 7 1 1 2 1 1 7 5 4 6 6 4 1 1 2 8 4 7 1 6 2 7 7 5 4 3 2 4 3 6 9 4 1 7 1 3 4 1 2 1 3 1 1 4 7 3 1 1 1 1 5 3 2 6 1 5 1 3 5 4 5 2 3 1 1 6 1 7 3 2 5 4 3 1 6 1 5 3 1 7 6 5 1 1 1 4 6 1 6 2 7 2 1 2 e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m m a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S HOXB6 homeo box B6 HOXB5 homeo box B5 WNT5A wingless-type MMTV integration site family, member 5A WNT5A wingless-type MMTV integration site family, member 5A FKBP11 FK506 binding protein 11, 19 kDa EPOR erythropoietin receptor SLC5A6 solute carrier family 5 sodium-dependent vitamin transporter, member 6 SLC5A6 solute carrier family 5 sodium-dependent vitamin transporter, member 6 RAD52 RAD52 homolog S.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al
    US 20030082511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown et al. (43) Pub. Date: May 1, 2003 (54) IDENTIFICATION OF MODULATORY Publication Classification MOLECULES USING INDUCIBLE PROMOTERS (51) Int. Cl." ............................... C12O 1/00; C12O 1/68 (52) U.S. Cl. ..................................................... 435/4; 435/6 (76) Inventors: Steven J. Brown, San Diego, CA (US); Damien J. Dunnington, San Diego, CA (US); Imran Clark, San Diego, CA (57) ABSTRACT (US) Correspondence Address: Methods for identifying an ion channel modulator, a target David B. Waller & Associates membrane receptor modulator molecule, and other modula 5677 Oberlin Drive tory molecules are disclosed, as well as cells and vectors for Suit 214 use in those methods. A polynucleotide encoding target is San Diego, CA 92121 (US) provided in a cell under control of an inducible promoter, and candidate modulatory molecules are contacted with the (21) Appl. No.: 09/965,201 cell after induction of the promoter to ascertain whether a change in a measurable physiological parameter occurs as a (22) Filed: Sep. 25, 2001 result of the candidate modulatory molecule. Patent Application Publication May 1, 2003 Sheet 1 of 8 US 2003/0082511 A1 KCNC1 cDNA F.G. 1 Patent Application Publication May 1, 2003 Sheet 2 of 8 US 2003/0082511 A1 49 - -9 G C EH H EH N t R M h so as se W M M MP N FIG.2 Patent Application Publication May 1, 2003 Sheet 3 of 8 US 2003/0082511 A1 FG. 3 Patent Application Publication May 1, 2003 Sheet 4 of 8 US 2003/0082511 A1 KCNC1 ITREXCHO KC 150 mM KC 2000000 so 100 mM induced Uninduced Steady state O 100 200 300 400 500 600 700 Time (seconds) FIG.
    [Show full text]
  • Supplementary Table 2
    Supplementary Table 2. Differentially Expressed Genes following Sham treatment relative to Untreated Controls Fold Change Accession Name Symbol 3 h 12 h NM_013121 CD28 antigen Cd28 12.82 BG665360 FMS-like tyrosine kinase 1 Flt1 9.63 NM_012701 Adrenergic receptor, beta 1 Adrb1 8.24 0.46 U20796 Nuclear receptor subfamily 1, group D, member 2 Nr1d2 7.22 NM_017116 Calpain 2 Capn2 6.41 BE097282 Guanine nucleotide binding protein, alpha 12 Gna12 6.21 NM_053328 Basic helix-loop-helix domain containing, class B2 Bhlhb2 5.79 NM_053831 Guanylate cyclase 2f Gucy2f 5.71 AW251703 Tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a 5.57 NM_021691 Twist homolog 2 (Drosophila) Twist2 5.42 NM_133550 Fc receptor, IgE, low affinity II, alpha polypeptide Fcer2a 4.93 NM_031120 Signal sequence receptor, gamma Ssr3 4.84 NM_053544 Secreted frizzled-related protein 4 Sfrp4 4.73 NM_053910 Pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 4.69 BE113233 Suppressor of cytokine signaling 2 Socs2 4.68 NM_053949 Potassium voltage-gated channel, subfamily H (eag- Kcnh2 4.60 related), member 2 NM_017305 Glutamate cysteine ligase, modifier subunit Gclm 4.59 NM_017309 Protein phospatase 3, regulatory subunit B, alpha Ppp3r1 4.54 isoform,type 1 NM_012765 5-hydroxytryptamine (serotonin) receptor 2C Htr2c 4.46 NM_017218 V-erb-b2 erythroblastic leukemia viral oncogene homolog Erbb3 4.42 3 (avian) AW918369 Zinc finger protein 191 Zfp191 4.38 NM_031034 Guanine nucleotide binding protein, alpha 12 Gna12 4.38 NM_017020 Interleukin 6 receptor Il6r 4.37 AJ002942
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • An NKX2-1/ERK/WNT Feedback Loop Modulates Gastric Identity And
    RESEARCH ARTICLE An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma Rediet Zewdu1,2, Elnaz Mirzaei Mehrabad1,3, Kelley Ingram1,4, Pengshu Fang1,4, Katherine L Gillis1,4, Soledad A Camolotto1,2, Grace Orstad1,4, Alex Jones1,2, Michelle C Mendoza1,4, Benjamin T Spike1,4, Eric L Snyder1,2,4* 1Huntsman Cancer Institute, Salt Lake City, United States; 2Department of Pathology, University of Utah, Salt Lake City, United States; 3School of Computing, University of Utah, Salt Lake City, United States; 4Department of Oncological Sciences, University of Utah, Salt Lake City, United States Abstract Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2- 1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.
    [Show full text]
  • To Nucleotide Sequence Determinations (Pancreatic Dnase I/U1 Ribonuclease/U2 Ribonuclease/Pancreatic Ribonuclease A/Ribosubstituted DNA) GARY V
    Proc. Nat. Acad. Sci. USA Vol. 71, No. 12, pp. 5017-5021, December 1974 Deoxysubstitution in RNA by RNA Polymerase In Vitro: A New Approach to Nucleotide Sequence Determinations (pancreatic DNase I/U1 ribonuclease/U2 ribonuclease/pancreatic ribonuclease A/ribosubstituted DNA) GARY V. PADDOCK, HOWARD C. HEINDELL AND WINSTON SALSER Biology Department and Molecular Biology Institute, University of California at Los Angeles, 405 Hilgard Ave., Los Angeles, Calif. 90024 Communicated by Charles Yanofsky, August 12, 1974 ABSTRACT Deoxynucleotides have been incorporated substituted RNA. We have observed that Mn++ ion will not into RNA synthesized in vitro by RNA polymerase with only cause DNA polymerase to synthesize ribosubstituted either double-stranded or single-stranded DNA as a to synthesize template. By use of this technique to block or promote DNA but will also cause RNA polymerase cleavage at a particular phosphodiester bond, a variety of deoxysubstituted RNA. Subsequently we have discovered a specific cleavages may be obtained with the available ribo- number of other papers dealing with the synthesis of deoxy- nucleases and deoxyribonuclease I. These methods should substituted RNA (12-14). These papers did not, however, greatly increase the ease and rapidity of nucleotide se- point out the applicability of the approach to nucleotide quence determinations. sequencing and did not include the controls we have found The introduction of radiographic approaches by Sanger and essential for demonstrating that complete deoxysubstitution his colleagues (1-4) greatly increased the power of nucleotide has occurred. sequencing techniques, but there remain certain obstacles In this preliminary paper we discuss the theory by which whose solution could result in impressive further increases in substitution of deoxynucleotides becomes an aid in nucleotide the rapidity with which large sequences may be determined.
    [Show full text]
  • Cell-Based Osteoprotegerin Therapy for Debris-Induced Aseptic Prosthetic Loosening on a Murine Model
    Gene Therapy (2010) 17, 1262–1269 & 2010 Macmillan Publishers Limited All rights reserved 0969-7128/10 www.nature.com/gt ORIGINAL ARTICLE Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model L Zhang1,2, T-H Jia2, ACM Chong1, L Bai1,HYu1, W Gong2, PH Wooley1 and S-Y Yang1 1Orthopaedic Research Institute, Via Christi Regional Medical Center, Wichita, KS, USA and 2Department of Orthopaedic Surgery, Jinan Central Hospital, Shandong University School of Medicine, Jinan, China Exogenous osteoprotegerin (OPG) gene modification membranes existed ubiquitously at bone–implant interface appears a therapeutic strategy for osteolytic aseptic in control groups, whereas only observed sporadically in OPG loosening. The feasibility and efficacy of a cell-based OPG gene-modified groups. Tartrate-resistant acid phosphatase+ gene delivery approach were investigated using a murine osteoclasts and tumor necrosis factor a,interleukin-1b, model of knee prosthesis failure. A titanium pin was CD68+ expressing cells were significantly reduced in implanted into mouse proximal tibia to mimic a weight- periprosthetic tissues of OPG gene-modified mice. No bearing knee arthroplasty, followed by titanium particles transgene dissemination or tumorigenesis was detected in challenge to induce periprosthetic osteolysis. Mouse fibro- remote organs and tissues. Data suggest that cell-based blast-like synoviocytes were transduced in vitro with either ex vivo OPG gene therapy was comparable in efficacy with AAV-OPG or AAV-LacZ before transfused into the osteolytic in vivo local gene transfer technique to deliver functional prosthetic joint 3 weeks post surgery. Successful transgene therapeutic OPG activities, effectively halted the debris- expression at the local site was confirmed 4 weeks later after induced osteolysis and regained the implant stability in this killing.
    [Show full text]
  • Figure S1. GO Analysis of Genes in Glioblastoma Cases That Showed Positive and Negative Correlations with TCIRG1 in the GSE16011 Cohort
    Figure S1. GO analysis of genes in glioblastoma cases that showed positive and negative correlations with TCIRG1 in the GSE16011 cohort. (A‑C) GO‑BP, GO‑CC and GO‑MF terms of genes that showed positive correlations with TCIRG1, respec‑ tively. (D‑F) GO‑BP, GO‑CC and GO‑MF terms of genes that showed negative correlations with TCIRG1. Red nodes represent gene counts, and black bars represent negative 1og10 P‑values. TCIRG1, T cell immune regulator 1; GO, Gene Ontology; BP, biological process; CC, cellular component; MF, molecular function. Table SI. Genes correlated with T cell immune regulator 1. Gene Name Pearson's r ARPC1B Actin‑related protein 2/3 complex subunit 1B 0.756 IL4R Interleukin 4 receptor 0.695 PLAUR Plasminogen activator, urokinase receptor 0.693 IFI30 IFI30, lysosomal thiol reductase 0.675 TNFAIP3 TNF α‑induced protein 3 0.675 RBM47 RNA binding motif protein 47 0.666 TYMP Thymidine phosphorylase 0.665 CEBPB CCAAT/enhancer binding protein β 0.663 MVP Major vault protein 0.660 BCL3 B‑cell CLL/lymphoma 3 0.657 LILRB3 Leukocyte immunoglobulin‑like receptor B3 0.656 ELF4 E74 like ETS transcription factor 4 0.652 ITGA5 Integrin subunit α 5 0.651 SLAMF8 SLAM family member 8 0.647 PTPN6 Protein tyrosine phosphatase, non‑receptor type 6 0.641 RAB27A RAB27A, member RAS oncogene family 0.64 S100A11 S100 calcium binding protein A11 0.639 CAST Calpastatin 0.638 EHBP1L1 EH domain‑binding protein 1‑like 1 0.638 LILRB2 Leukocyte immunoglobulin‑like receptor B2 0.629 ALDH3B1 Aldehyde dehydrogenase 3 family member B1 0.626 GNA15 G protein
    [Show full text]
  • Deoxyribonuclease I
    Catalog Number: 100574, 100575, 190062 Deoxyribonuclease I CAS #: 9003-98-9 Description: Deoxyribonuclease from beef pancreas, DNase I, was first crystallized by Kunitz.10,11 It is an endonuclease which splits phosphodiester linkages, preferentially adjacent to a pyrimidine nucleotide yielding 5'-phosphate terminated polynucleotides with a free hydroxyl group on position 3'. The average chain of limit digest is a tetranucleotide.19 DNase I acts upon single chain DNA29, and upon double-stranded DNA and chromatin. In the latter case, although histones restrict susceptibility to nuclease action, over a period of time nearly all chromatin DNA is acted upon. According to Mirsky and Silverman20, this could result from the looseness of histone attachment to DNA. They found that lysine-rich histones more effectively block DNase access to DNA than arginine-rich histones. Billing and Bonner2 suggest that DNase attacks the histone-free strand of chromatin DNA. Schmidt, et. al.28 indicate that hydrolysis of the histone-free region of DNA strands accounts for the initial rapid action of the enzyme on chromatin. Bollum3 reports degradation of synthetic homopolymer complexes by DNase I. The intracellular functions of the enzyme are probably controlled by a DNase inhibitor17, which according to Lazarides and Lindberg13 is actin. Molecular weight: 31,000.18 Composition: There are four deoxyribonucleases of beef pancreas: A, B, C, and D.14, 25, 27 Five have been reported by Junowicz and Spencer.7 They are glycoproteins differing from each other either in carbohydrate side-chain or polypeptide component.14, 25 DNase A is the predominant form26; its amino acid sequence has been reported.16 Optimum pH: 7.8.
    [Show full text]
  • Skeletal Stem and Progenitor Cells Maintain Cranial Suture Patency and Prevent Craniosynostosis
    ARTICLE https://doi.org/10.1038/s41467-021-24801-6 OPEN Skeletal stem and progenitor cells maintain cranial suture patency and prevent craniosynostosis Siddharth Menon1,2,3,4, Ankit Salhotra 1,2,3, Siny Shailendra1,2,3, Ruth Tevlin1,2,3, Ryan C. Ransom1,2,3, Michael Januszyk1,2,3, Charles K. F. Chan 1,2,3,4, Björn Behr5, Derrick C. Wan1,2,3, ✉ ✉ Michael T. Longaker 1,2,3,4 & Natalina Quarto 1,2,3,6 Cranial sutures are major growth centers for the calvarial vault, and their premature fusion 1234567890():,; leads to a pathologic condition called craniosynostosis. This study investigates whether skeletal stem/progenitor cells are resident in the cranial sutures. Prospective isolation by FACS identifies this population with a significant difference in spatio-temporal representation between fusing versus patent sutures. Transcriptomic analysis highlights a distinct signature in cells derived from the physiological closing PF suture, and scRNA sequencing identifies transcriptional heterogeneity among sutures. Wnt-signaling activation increases skeletal stem/progenitor cells in sutures, whereas its inhibition decreases. Crossing Axin2LacZ/+ mouse, endowing enhanced Wnt activation, to a Twist1+/− mouse model of coronal cra- niosynostosis enriches skeletal stem/progenitor cells in sutures restoring patency. Co- transplantation of these cells with Wnt3a prevents resynostosis following suturectomy in Twist1+/− mice. Our study reveals that decrease and/or imbalance of skeletal stem/pro- genitor cells representation within sutures may underlie craniosynostosis. These findings have translational implications toward therapeutic approaches for craniosynostosis. 1 Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA. 2 Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, CA, USA.
    [Show full text]
  • Deoxyribonuclease I, Bovine (D2821)
    Deoxyribonuclease I, bovine recombinant, expressed in Pichia pastoris lyophilized powder Catalog Number D2821 Storage Temperature 2–8 °C CAS RN 9003-98-9 Optimal pH: EC 3.1.21.1 The optimal pH of DNase I activity is dependent on the Synonyms: DNase I, divalent ion present. In the presence of both Mg2+ and Deoxyribonucleate 5¢-Oligonucleotidohydrolase Ca2+, the optimal pH is between 7–8, while in the absence of Ca2+, the optimal pH is between 5.5–6.0.6 Product Description 1% Deoxyribonuclease I (DNase I) is found in most cells Extinction Coefficient: E280 = 11.1 and tissues. In mammals, the pancreas is one of the best sources for the enzyme. Pancreatic DNase I was This recombinant bovine DNase I is a glycoprotein, the first isolated DNase. produced without the addition of any animal-derived materials. It is supplied as a lyophilized powder DNase I is an endonuclease that acts on containing a glycine stabilizer. phosphodiester bonds adjacent to pyrimidines to produce polynucleotides with terminal 5¢-phosphates. Molecular mass: ~39 kDa A tetranucleotide is the smallest average digestion product. DNase I hydrolyzes single- and double- Specific activity: ³4,000 units/mg protein stranded DNA. In the presence of Mg2+ ions, DNase I attacks each strand of DNA independently and the Unit definition: One unit will produce a change in A260 of cleavage sites are random. If Mn2+ ions are present, 0.001 per minute per ml at pH 5.0 at 25 °C using DNA, both DNA strands are cleaved at approximately the Type I or III, as the substrate.
    [Show full text]