Protein and messenger RNA expression of connexin43 in astrocytomas: implications in brain tumor gene therapy

Restricted access

✓ The expression of connexin43, the primary gap-junction constituent of glial cells, was evaluated at the messenger RNA and protein levels in different grades of astrocytoma to investigate the relevance of gap junctions in herpes simplex virus—thymidine kinase (HSV-tk)—mediated gene therapy of brain tumors. Transduction of the retroviral-mediated HSV-tk gene into tumor cells with subsequent administration of ganciclovir has recently been used as an experimental therapeutic strategy for treatment of brain tumors. One aspect of this approach is the bystander effect, which augments the efficacy of this therapeutic approach. Glioblastoma cells with minimum levels of connexin43 protein were transfected with a connexin43 complementary DNA. These cells manifested a marked increase in the in vitro bystander effect, supporting the contention that the in vitro bystander effect is a consequence of metabolic cooperation between cells mediated by gap junctions. To assess relative levels of gap-junction protein expression in the relevant tumor type, we examined primary astrocytomas, primary astrocytoma cell cultures, and glioblastoma cell lines. Although most astrocytoma tumor samples expressed connexin43, they differed in the level of expression, with the greatest variation exhibited in high-grade astrocytomas. Primary glioblastoma cell cultures and established glioblastoma cell lines also displayed some variability in connexin43 levels. In aggregate, our results anticipate that glioblastomas will have a varied bystander effect during HSV-tk gene therapy depending on the level of connexin43 expression.

Article Information

Contributor Notes

Address reprint requests to: Peter J. Stambrook, Ph.D., Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati Medical Center, Cincinnati, Ohio 45267–0521.

© AANS, except where prohibited by US copyright law.

Headings
References
  • 1.

    Beyer ECPaul DLGoodenough DA: Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:262126291987Beyer EC Paul DL Goodenough DA: Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol 105:2621–2629 1987

    • Search Google Scholar
    • Export Citation
  • 2.

    Bi WLParysek LMWarnick Ret al: In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy. Hum Gene Ther 4:7257311993Bi WL Parysek LM Warnick R et al: In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy. Hum Gene Ther 4:725–731 1993

    • Search Google Scholar
    • Export Citation
  • 3.

    Chomczynski PSacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:1561591987Chomczynski P Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159 1987

    • Search Google Scholar
    • Export Citation
  • 4.

    Culver KWRam ZWallbridge Set al: In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256:155015521992Culver KW Ram Z Wallbridge S et al: In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 256:1550–1552 1992

    • Search Google Scholar
    • Export Citation
  • 5.

    Culver KWVan Gilder JLink CJet al: Gene therapy for the treatment of malignant brain tumors with in vivo tumor transduction with the herpes simplex thymidine kinase gene/ganciclovir system. Hum Gene Ther 5:3433791994Culver KW Van Gilder J Link CJ et al: Gene therapy for the treatment of malignant brain tumors with in vivo tumor transduction with the herpes simplex thymidine kinase gene/ganciclovir system. Hum Gene Ther 5:343–379 1994

    • Search Google Scholar
    • Export Citation
  • 6.

    Dermietzel RHertzberg ELKessler JAet al: Gap junctions between cultured astrocytes: immunocytochemical, molecular, and electrophysiological analysis. J Neurosci 11:142114231991Dermietzel R Hertzberg EL Kessler JA et al: Gap junctions between cultured astrocytes: immunocytochemical molecular and electrophysiological analysis. J Neurosci 11:1421–1423 1991

    • Search Google Scholar
    • Export Citation
  • 7.

    Dermietzel RTraub OHwang TKet al: Differential expression of three gap junction proteins in developing and mature brain tissues. Proc Natl Acad Sci USA 86:10148101521989Dermietzel R Traub O Hwang TK et al: Differential expression of three gap junction proteins in developing and mature brain tissues. Proc Natl Acad Sci USA 86:10148–10152 1989

    • Search Google Scholar
    • Export Citation
  • 8.

    Eghbali BKessler JAReid LMet al: Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin 32 cDNA retards growth in vivo. Proc Natl Acad Sci USA 88:10701107051991Eghbali B Kessler JA Reid LM et al: Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin 32 cDNA retards growth in vivo. Proc Natl Acad Sci USA 88:10701–10705 1991

    • Search Google Scholar
    • Export Citation
  • 9.

    El-Sabban MEPauli BU: Cytoplasmic dye transfer between metastatic tumor cells and vascular endothelium. J Cell Biol 115:137513821991El-Sabban ME Pauli BU: Cytoplasmic dye transfer between metastatic tumor cells and vascular endothelium. J Cell Biol 115:1375–1382 1991

    • Search Google Scholar
    • Export Citation
  • 10.

    Freeman SMAbboud CNWhartenby KAet al: The “bystander effect”: tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 53:527452831993Freeman SM Abboud CN Whartenby KA et al: The “bystander effect”: tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 53:5274–5283 1993

    • Search Google Scholar
    • Export Citation
  • 11.

    Giaume CFromaget Cel Aoumari Aet al: Gap junctions in cultured astrocytes: single-channel currents and characterization of channel-forming protein. Neuron 6:1331431991Giaume C Fromaget C el Aoumari A et al: Gap junctions in cultured astrocytes: single-channel currents and characterization of channel-forming protein. Neuron 6:133–143 1991

    • Search Google Scholar
    • Export Citation
  • 12.

    Gross JLBehrens DLMullins DEet al: Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate. Cancer Res 48:2912961988Gross JL Behrens DL Mullins DE et al: Plasminogen activator and inhibitor activity in human glioma cells and modulation by sodium butyrate. Cancer Res 48:291–296 1988

    • Search Google Scholar
    • Export Citation
  • 13.

    Hendrix EMMao SJTEverson Wet al: Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor. Mol Reprod Dev 33:27381992Hendrix EM Mao SJT Everson W et al: Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor. Mol Reprod Dev 33:27–38 1992

    • Search Google Scholar
    • Export Citation
  • 14.

    Hossain MZMurphy LJHertzberg ELet al: Phosphorylated forms of connexin43 predominate in rat brain: demonstration by rapid inactivation of brain metabolism. J Neurochem 62:239424031994Hossain MZ Murphy LJ Hertzberg EL et al: Phosphorylated forms of connexin43 predominate in rat brain: demonstration by rapid inactivation of brain metabolism. J Neurochem 62:2394–2403 1994

    • Search Google Scholar
    • Export Citation
  • 15.

    Kleihues PBurger PCScheithauer BW: The new WHO classification of brain tumours. Brain Pathol 3:2552681993Kleihues P Burger PC Scheithauer BW: The new WHO classification of brain tumours. Brain Pathol 3:255–268 1993

    • Search Google Scholar
    • Export Citation
  • 16.

    Laird DWPuranam KLRevel JP: Identification of intermediate forms of connexin43 in rat cardiac myocytes in Hall JEZampigh GADavis RM (eds): Gap Junctions. Amsterdam: Elsevier1993 pp 263268Laird DW Puranam KL Revel JP: Identification of intermediate forms of connexin43 in rat cardiac myocytes in Hall JE Zampigh GA Davis RM (eds): Gap Junctions. Amsterdam: Elsevier 1993 pp 263–268

    • Search Google Scholar
    • Export Citation
  • 17.

    Laird DWPuranam KLRevel JP: Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes. Biochem J 273:67721991Laird DW Puranam KL Revel JP: Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes. Biochem J 273:67–72 1991

    • Search Google Scholar
    • Export Citation
  • 18.

    Larson DMHaudenschild CCBeyer EC: Gap junction messenger RNA expression by vascular wall cells. Circ Res 66:107410801990Larson DM Haudenschild CC Beyer EC: Gap junction messenger RNA expression by vascular wall cells. Circ Res 66:1074–1080 1990

    • Search Google Scholar
    • Export Citation
  • 19.

    Loewenstein WRKanno Y: Intercellular communication and the control of tissue growth: lack of communication between cancer cells. Nature 209:124812491966Loewenstein WR Kanno Y: Intercellular communication and the control of tissue growth: lack of communication between cancer cells. Nature 209:1248–1249 1966

    • Search Google Scholar
    • Export Citation
  • 20.

    Loewenstein WRKanno Y: Studies on an epithelial (gland) cell junction. 1. Modifications of surface membrane permeability. J Cell Biol 22:5655861964Loewenstein WR Kanno Y: Studies on an epithelial (gland) cell junction. 1. Modifications of surface membrane permeability. J Cell Biol 22:565–586 1964

    • Search Google Scholar
    • Export Citation
  • 21.

    Mehta PPBertram JSLoewenstein WR: Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell 44:1871961986Mehta PP Bertram JS Loewenstein WR: Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell 44:187–196 1986

    • Search Google Scholar
    • Export Citation
  • 22.

    Mehta PPHotz-Wagenblatt ARose Bet al: Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth. J Membr Biol 124:2072251991Mehta PP Hotz-Wagenblatt A Rose B et al: Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth. J Membr Biol 124:207–225 1991

    • Search Google Scholar
    • Export Citation
  • 23.

    Micevych PEAbelson L: Distribution of mRNAs coding for liver and heart gap junction proteins in the rat central nervous system. J Comp Neurol 305:961181991Micevych PE Abelson L: Distribution of mRNAs coding for liver and heart gap junction proteins in the rat central nervous system. J Comp Neurol 305:96–118 1991

    • Search Google Scholar
    • Export Citation
  • 24.

    Moolten FL: Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 46:527652811986Moolten FL: Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 46:5276–5281 1986

    • Search Google Scholar
    • Export Citation
  • 25.

    Morgenstern JPLand H: Advanced mammalian gene transfer: high titer retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18:357835961990Morgenstern JP Land H: Advanced mammalian gene transfer: high titer retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18:3578–3596 1990

    • Search Google Scholar
    • Export Citation
  • 26.

    Musil LSCunningham BAEdelman GMet al: Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J Cell Biol 111:207720881990Musil LS Cunningham BA Edelman GM et al: Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J Cell Biol 111:2077–2088 1990

    • Search Google Scholar
    • Export Citation
  • 27.

    Musil LSGoodenough DA: Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell 74:106510771993Musil LS Goodenough DA: Multisubunit assembly of an integral plasma membrane channel protein gap junction connexin43 occurs after exit from the ER. Cell 74:1065–1077 1993

    • Search Google Scholar
    • Export Citation
  • 28.

    Naus CCGElisevich KZhu Det al: In vivo growth of C6 glioma cells transfected with connexin43 cDNA. Cancer Res 52:420842131992Naus CCG Elisevich K Zhu D et al: In vivo growth of C6 glioma cells transfected with connexin43 cDNA. Cancer Res 52:4208–4213 1992

    • Search Google Scholar
    • Export Citation
  • 29.

    Nicholson BDermietzel RTeplow Det al: Two homologous protein components of hepatic gap junctions. Nature 329:7327341987Nicholson B Dermietzel R Teplow D et al: Two homologous protein components of hepatic gap junctions. Nature 329:732–734 1987

    • Search Google Scholar
    • Export Citation
  • 30.

    Nicolson GLDulski KMTrosko JE: Loss of intercellular junctional communication correlates with metastatic potential in mammary adenocarcinoma cells. Proc Natl Acad Sci USA 85:4734761988Nicolson GL Dulski KM Trosko JE: Loss of intercellular junctional communication correlates with metastatic potential in mammary adenocarcinoma cells. Proc Natl Acad Sci USA 85:473–476 1988

    • Search Google Scholar
    • Export Citation
  • 31.

    Owens RBSmith HSNelson-Rees WAet al: Epithelial cell cultures from normal and cancerous human tissues. J Natl Cancer Inst 56:8438491976Owens RB Smith HS Nelson-Rees WA et al: Epithelial cell cultures from normal and cancerous human tissues. J Natl Cancer Inst 56:843–849 1976

    • Search Google Scholar
    • Export Citation
  • 32.

    Pontén JMacintyre EH: Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand 74:4654861968Pontén J Macintyre EH: Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol Scand 74:465–486 1968

    • Search Google Scholar
    • Export Citation
  • 33.

    Puranam KLLaird DWRevel JP: Trapping an intermediate form of connexin43 in the golgi. Exp Cell Res 206:85921993Puranam KL Laird DW Revel JP: Trapping an intermediate form of connexin43 in the golgi. Exp Cell Res 206:85–92 1993

    • Search Google Scholar
    • Export Citation
  • 34.

    Rose BMehta PPLoewenstein WR: Gap-junction protein gene suppresses tumorigenicity. Carcinogenesis 14:107310751993Rose B Mehta PP Loewenstein WR: Gap-junction protein gene suppresses tumorigenicity. Carcinogenesis 14:1073–1075 1993

    • Search Google Scholar
    • Export Citation
  • 35.

    Selden RF: Analysis of RNA by Northern hybridization in Ausubel FMBrent RKingston REet al (eds): Current Protocols in Molecular Biologyed 2. New York: John Wiley & Sons1992 pp 4-234-25Selden RF: Analysis of RNA by Northern hybridization in Ausubel FM Brent R Kingston RE et al (eds): Current Protocols in Molecular Biology ed 2. New York: John Wiley & Sons 1992 pp 4-23–4-25

    • Search Google Scholar
    • Export Citation
  • 36.

    Stein GH: T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro. J Cell Physiol 99:43541979Stein GH: T98G: an anchorage-independent human tumor cell line that exhibits stationary phase G1 arrest in vitro. J Cell Physiol 99:43–54 1979

    • Search Google Scholar
    • Export Citation
  • 37.

    Traub OLook JDermietzel Ret al: Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes. J Cell Biol 108:103910511989Traub O Look J Dermietzel R et al: Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes. J Cell Biol 108:1039–1051 1989

    • Search Google Scholar
    • Export Citation
  • 38.

    Wilgenbus KKKirkpatrick CJKnuechel Ret al: Expression of Cx26, Cx32 and Cx43 gap junction proteins in normal and neoplastic human tissues. Int J Cancer 51:5225291992Wilgenbus KK Kirkpatrick CJ Knuechel R et al: Expression of Cx26 Cx32 and Cx43 gap junction proteins in normal and neoplastic human tissues. Int J Cancer 51:522–529 1992

    • Search Google Scholar
    • Export Citation
  • 39.

    Yamamoto TOchalski AHertzberg ELet al: On the organization of astrocytic gap junctions in rat brain as suggested by LM and EM immunohistochemistry of connexin43 expression. J Comp Neurol 302:8538831990Yamamoto T Ochalski A Hertzberg EL et al: On the organization of astrocytic gap junctions in rat brain as suggested by LM and EM immunohistochemistry of connexin43 expression. J Comp Neurol 302:853–883 1990

    • Search Google Scholar
    • Export Citation
  • 40.

    Yamamoto TShiosaka SWhittaker MEet al: Gap junction protein in rat hippocampus: light microscope immunohistochemical localization. J Comp Neurol 281:2692811989Yamamoto T Shiosaka S Whittaker ME et al: Gap junction protein in rat hippocampus: light microscope immunohistochemical localization. J Comp Neurol 281:269–281 1989

    • Search Google Scholar
    • Export Citation
  • 41.

    Zhu DCaveney SKidder GMet al: Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation. Proc Natl Acad Sci USA 88:188318871991Zhu D Caveney S Kidder GM et al: Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression intercellular coupling and cell proliferation. Proc Natl Acad Sci USA 88:1883–1887 1991

    • Search Google Scholar
    • Export Citation
TrendMD
Cited By
Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 251 248 98
Full Text Views 154 82 0
PDF Downloads 54 31 0
EPUB Downloads 0 0 0
PubMed
Google Scholar