The Journal of Membrane Biology

, Volume 124, Issue 3, pp 207–225 | Cite as

Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth

  • Parmender P. Mehta
  • Agnes Hotz-Wagenblatt
  • Birgit Rose
  • David Shalloway
  • Warner R. Loewenstein
Articles

Summary

Incorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals.

Key Words

intercellular communication gap junction connexin growth control cDNA connexin43 cell-cell channel junctional communication transformation cancer etiology 

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References

  1. Atkinson, M.M., Menko, A.S., Johnson, R.G., Sheppard, J.R., Sheridan, J.D. 1981. Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus.J. Cell Biol. 91:573–578PubMedGoogle Scholar
  2. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. 1987. Current Protocols in Molecular Biology. John Wiley & Sons, New YorkGoogle Scholar
  3. Azarnia, R., Loewenstein, W.R. 1984. Intercellular communication and the control of growth: X. Alteration of junctional permeability by thesrc gene. A study with temperaturesensitive mutant Rous sarcoma virus.J. Membrane Biol. 82:191–205Google Scholar
  4. Azarnia, R., Loewenstein, W.R. 1987. Polyomavirus middle T antigen downregulates junctional cell-to-cell communication.Mol. Cell. Biol. 7:946–950PubMedGoogle Scholar
  5. Azarnia, R., Mitcho, M., Shalloway, D., Loewenstein, W.R. 1989. Junctional intercellular communication is cooperatively inhibited by oncogenes in transformation.Oncogene 4:1161–1168PubMedGoogle Scholar
  6. Azarnia, R., Reddy, S., Kmiecik, T.E., Shalloway, D., Loewenstein, W.R. 1988. The cellularsrc gene product regulates junctional cell-to-cell communication.Science 239:398–401PubMedGoogle Scholar
  7. Beyer, E.C., Kistler, J., Paul, D.L., Goodenough, D.A. 1989. Antisera directed against connexin 43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues.J. Cell. Biol. 108:595–605PubMedGoogle Scholar
  8. Beyer, E.C., Paul, D.L., Goodenough, D.A. 1987. Connexin 43: A protein from rat heart homologous to a gap junction protein from liver.J. Cell Biol. 105:2621–2629PubMedGoogle Scholar
  9. Borek, C., Higashino, S., Loewenstein, W.R. 1969. Intercellular communication and tissue growth: IV. Conductance of membrane junctions of normal and cancerous cells in culture.J. Membrane Biol. 1:274–293Google Scholar
  10. Boulter, C.A., Wagner, E.F. 1987. A universal retroviral vector for efficient constitutive expression of exogenous genes.Nucleic Acids Res. 15:7194PubMedGoogle Scholar
  11. Chang, C.C., Trosko, J.E., Kung, H.J., Bombick, D., Matsumura, F. 1985. Potential role of thesrc gene product in inhibition of gap-junctional communication in NIH/3T3 cells.Proc. Natl. Acad. Sci. USA 82:5360–5364PubMedGoogle Scholar
  12. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry 18:5294–5299PubMedGoogle Scholar
  13. Chomezynski, P., Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.Anal. Biochem. 162:156–159PubMedGoogle Scholar
  14. Crow, D.S., Beyer, E.C., Paul, D.L., Kobe, S.S., Lau, A.F. 1990. Phosphorylation of connexin43 gap junction protein in uninfected and rous sarcoma virus-transformed mammalian fibroblasts.Mol. Cell. Biol. 10:1754–1763PubMedGoogle Scholar
  15. Eghbali, B., Kessler, J.A., Spray, D.C. 1990. Expression of gap junction channels in communication-incompetent cells after stable transfection with cDNA encoding connexin 32.Proc. Natl. Acad. Sci. USA 87:1328–1331PubMedGoogle Scholar
  16. Enomoto, T., Sasaki, Y., Shiba, Y., Kanno, Y., Yamasaki, H. 1981. Tumor promotors cause a rapid and reversible inhibition of the formation and maintenance of electrical cell coupling in culture.Proc. Natl. Acad. Sci. USA 78:5628–5632PubMedGoogle Scholar
  17. Filson, A.J., Azarnia, R., Beyer, E.C., Loewenstein, W.R., Brugge, J.S. 1990. Tyrosine phosphorylation correlates with inhibition of cell-to-cell communication.Cell Growth Diff. 1:661–668PubMedGoogle Scholar
  18. Flagg-Newton, J.L., Simpson, I., Loewenstein, W.R. 1979. Permeability of the cell-to-cell membrane channels in mammalian cell junction.Science 205:404–407PubMedGoogle Scholar
  19. Gimlich, R.L., Kumar, N.M., Gilula, N.B. 1990. Differential regulation of the levels of three gap junction mRNAs inXenopus embryos.J. Cell Biol. 110:597–605PubMedGoogle Scholar
  20. Green, S., Issemann, I., Sheer, E. 1988. A versatilein vivo andin vitro eukaryotic expression vector for protein engineering.Nucleic Acids Res. 16:369PubMedGoogle Scholar
  21. Guthrie, S.C., Gilula, N.B. 1989. Gap junctional communication and development.Trends Neurosci. 12:12–16PubMedGoogle Scholar
  22. Hantzopoulos, P.A., Sullinger, B.A., Ungers, G., Gilboa, E. 1989. Improved gene expression upon transfer of adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.Proc. Natl. Acad. Sci. USA 86:3519–3523PubMedGoogle Scholar
  23. Hoh, J.H., John, S.A., Revel, J.-P. 1991. Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31.J. Biol. Chem. 266:6524–6531PubMedGoogle Scholar
  24. Honig, M.G., Hume, R.I. 1986. Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.J. Cell Biol. 103:171–187PubMedGoogle Scholar
  25. Korman, A.J., Frantz, D., Strominger, J.L., Mulligan, R.C. 1987. Expression of human class II major histocompatibility complex antigens using retrovirus vectors.Proc. Natl. Acad. Sci. USA 84:2150–2154PubMedGoogle Scholar
  26. Kumar, N.M., Gilula, N.B. 1986. Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein.J. Cell Biol. 103:767–776PubMedGoogle Scholar
  27. Lee, S.W., Tomasetto, C., Sager, R. 1991. Positive selection of candidate tumor-suppressor genes by subtractive hybridization.Proc. Natl. Acad. Sci. USA 88:2825–2829PubMedGoogle Scholar
  28. Lehrach, H.D., Diamond, D., Wozney, J.M., Boedtker, H. 1977. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination.Biochemistry 16:4743–4751PubMedGoogle Scholar
  29. Loewenstein, W.R. 1966. Permeability of membrane junctions.Ann. N.Y. Acad. Sci. 137:441–472PubMedGoogle Scholar
  30. Loewenstein, W.R. 1967. On the genesis of cellular communication.Dev. Biol. 15:503–520PubMedGoogle Scholar
  31. Loewenstein, W.R. 1979. Junctional intercellular communication and the control of growth.Biochim. Biophys. Acta Cancer Rev. 560:1–65Google Scholar
  32. Loewenstein, W.R. 1981. Junctional intercellular communication. The cell-to-cell membrane channel.Physiol. Rev. 61:829–913PubMedGoogle Scholar
  33. MacGregor, G.R., Caskey, C.T. 1989. Constructions of plasmids that expressE. coli β-galactosidase in mammalian cells.Nucleic Acids Res. 17:2365PubMedGoogle Scholar
  34. Maldonado, P.E., Rose, B., Loewenstein, W.R. 1988. Growth factors modulate junctional cell-to-cell communication.J. Membrane Biol. 106:203–210Google Scholar
  35. Maniatis, T., Fritsch, E.F., Sambrook, J. 1989. Molecular Cloning: A Laboratory Manual. Vol. 1–3. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  36. Mehta, P.P., Bertram, J.S., Loewenstein, W.R. 1986. Growth inhibition of transformed cells correlates with their junctional communication with normal cells.Cell 44:187–196PubMedGoogle Scholar
  37. Mehta, P.P., Bertram, J.S., Loewenstein, W.R. 1989. The actions of retinoids on cellular growth correlate with their actions on gap junctional communication.J. Cell Biol. 108:1053–1065PubMedGoogle Scholar
  38. Mehta, P.P., Loewenstein, W.R. 1991. Differential regulation of communication by retinoic acid in homologous and heterologous junctions between normal and transformed cells.J. Cell Biol. 113:371–379PubMedGoogle Scholar
  39. Miller, A.D., Buttimore, C. 1986. Redesign of retrovirus packaging cell lines to avoid recombination leading to Helper virus production.Mol. Cell. Biol. 6:2895–2902PubMedGoogle Scholar
  40. Murray, A.W., Fitzgerald, D.J. 1979. Tumor promotors inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells.Biochem. Biophys. Res. Commun. 91:395–401PubMedGoogle Scholar
  41. Musil, L.S., Beyer, E.C., Goodenough, D.A. 1990a. Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning, ultrastructural localization, and post-translational phosphorylation.J. Membrane Biol. 116:163–175Google Scholar
  42. Musil, L.S., Cunningham, B.A., Edelman, G.M., Goodenough, D.A. 1990b. Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and-deficient cell lines.J. Cell Biol. 111:2077–2088PubMedGoogle Scholar
  43. Nonner, W.F., Loewenstein, W.R. 1989. Appendix: A growth control model with discrete regulatory centers.J. Cell Biol. 108:1063–1065Google Scholar
  44. Paul, D.L. 1986. Molecular cloning cDNA for rat liver gap junction protein.J. Cell Biol. 103:123–134PubMedGoogle Scholar
  45. Reznikoff, C.A., Bertram, J.S., Brankow, D.W., Heidelberger, C. 1973. Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence inhibition of cell division.Cancer Res. 33:3239–3249PubMedGoogle Scholar
  46. Rogers, M., Berestecky, J.M., Hossain, M.Z., Guo, H., Kadle, R., Nicholson, B., Bertram, J.S. 1990. Retinoid-enhanced gap junctional communication is achieved by increased levels of connexin43 mRNA and protein.Mol. Carcinogen. 3:335–343Google Scholar
  47. Rosen, A., Van Der Merwe, P.A., Davidson, J.S. 1988. Effects of SV40 transformation on intercellular gap junctional communication in human fibroblasts.Cancer Res. 48:3485–3489PubMedGoogle Scholar
  48. Schwartzmann, G.O.H., Wiegandt, H., Rose, B., Zimmerman, A., Ben-Haim, D., Loewenstein, W.R. 1981. The diameter of the cell-to-cell junctional membrane channels, as probed with neutral molecules.Science 213:551–553PubMedGoogle Scholar
  49. Swenson, K.I., Piwnica-Worms, H., McNamee, H., Paul, D.L. 1990. Tyrosine phosphorylation of the gap junction protein connexin 43 accounts for the pp60v-src-induced inhibition of communication inXenopus oocyte pairs.Cell Reg. 1(13):989–1002Google Scholar
  50. Thomas, K.R., Capecchi, M.R. 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.Cell 51:503–512PubMedGoogle Scholar
  51. Tso, J.Y., Sun, X.-H., Kao, T.-H., Reece, K.S., Wu, R. 1985. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: Genomic complexity and molecular evolution of the gene.Nucleic Acids Res. 13:2485–2502PubMedGoogle Scholar
  52. Unwin, P.N.T., Zampighi, G. 1980. Structure of the junction between communicating cells.Nature 283:545–549PubMedGoogle Scholar
  53. Yotti, L.P., Chang, C.C., Trosko, J.E. 1979. Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter.Science 206:1089–1091PubMedGoogle Scholar
  54. Zhang, J.-T., Nicholson, B.J. 1989. Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA.J. Cell Biol. 109:3391–3401PubMedGoogle Scholar
  55. Zhu, D., Caveney, S., Kidder, G.M., Naus, C.C.G. 1991. Transfection of C6 glioma cells with connexin 43 cDNA: Analysis of expression, intercellular coupling, and cell proliferation.Proc. Natl. Acad. Sci. USA 88:1883–1887PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 1991

Authors and Affiliations

  • Parmender P. Mehta
    • 1
  • Agnes Hotz-Wagenblatt
    • 2
  • Birgit Rose
    • 1
  • David Shalloway
    • 2
  • Warner R. Loewenstein
    • 1
  1. 1.Department of Physiology and BiophysicsUniversity of Miami School of MedicineMiami
  2. 2.Section of Biochemistry, Molecular and Cell Biology and Department of PathologyCornell UniversityIthaca

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