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. 1986 Aug 1;103(2):361–367. doi: 10.1083/jcb.103.2.361

Contact inhibition of growth of human diploid fibroblasts by immobilized plasma membrane glycoproteins

PMCID: PMC2113841  PMID: 3733871

Abstract

The human embryonic fibroblasts used in this study show pronounced inhibition of growth when reaching a critical cell density. High cell density and growth inhibition has previously been mimicked by the addition of glutaraldehyde-fixed cells or of isolated plasma membranes to sparsely seeded proliferating fibroblasts (Wieser, R. J., R. Heck, and F. Oesch, 1985, Exp. Cell Res., 158:493-499). In this report, we describe the successful solubilization of the growth-inhibiting glycoproteins and their covalent coupling to silicabeads (10 microns), which had been derivatized with 3-isothiocyanatopropyltriethoxysilane. The beads, bearing the plasma membrane proteins, were added to sparsely seeded, actively proliferating fibroblasts, and growth was measured by the determination of cell number or of incorporation of [3H]thymidine into DNA. The growth was inhibited in a concentration-dependent manner, whereby 50% inhibition was achieved with 0.3 micrograms of immobilized protein added to 5 X 10(3) cells. Terminal galactose residues of plasma membrane glycoproteins with N-glycosydically bound carbohydrates were responsible for the inhibition of growth. Dense cultures of human fibroblasts are characterized by an accelerated synthesis of procollagen type III. We have found that this cellular response can also be induced by the addition of immobilized plasma membrane glycoproteins to sparsely seeded cells. These observations support the conclusion that the addition of immobilized plasma membrane glycoproteins to sparsely seeded fibroblasts mimics the situation occurring at high cell density. These results show that cell-cell contacts via plasma membrane glycoproteins carrying terminal galactose residues are important for the regulation of the proliferation of cultured human fibroblasts and presumably of the accelerated synthesis of collagen type III.

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Selected References

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  1. Abe S., Steinmann B. U., Wahl L. M., Martin G. R. High cell density alters the ratio of type III to I collagen synthesis by fibroblasts. Nature. 1979 May 31;279(5712):442–444. doi: 10.1038/279442a0. [DOI] [PubMed] [Google Scholar]
  2. Elbein A. D. Inhibitors of the biosynthesis and processing of N-linked oligosaccharides. CRC Crit Rev Biochem. 1984;16(1):21–49. doi: 10.3109/10409238409102805. [DOI] [PubMed] [Google Scholar]
  3. Fritze L. M., Reilly C. F., Rosenberg R. D. An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells. J Cell Biol. 1985 Apr;100(4):1041–1049. doi: 10.1083/jcb.100.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gotlib L. J. Isolation of cell plasma membranes on microcarrier culture beads. Biochim Biophys Acta. 1982 Feb 8;685(1):21–26. doi: 10.1016/0005-2736(82)90029-3. [DOI] [PubMed] [Google Scholar]
  5. Hjelmeland L. M. A nondenaturing zwitterionic detergent for membrane biochemistry: design and synthesis. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6368–6370. doi: 10.1073/pnas.77.11.6368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Holley R. W., Kiernan J. A. "Contact inhibition" of cell division in 3T3 cells. Proc Natl Acad Sci U S A. 1968 May;60(1):300–304. doi: 10.1073/pnas.60.1.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hsu Y. M., Barry J. M., Wang J. L. Growth control in cultured 3T3 fibroblasts: neutralization and identification of a growth-inhibitory factor by a monoclonal antibody. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2107–2111. doi: 10.1073/pnas.81.7.2107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kinders R. J., Milenkovic A. G., Nordin P., Johnson T. C. Characterization of cell-surface glycopeptides from mouse cerebral cortex that inhibit cell growth and protein synthesis. Biochem J. 1980 Sep 15;190(3):605–614. doi: 10.1042/bj1900605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kinkel J. N., Anspach B., Unger K. K., Wieser R., Brunner G. Separation of plasma membrane proteins of cultured human fibroblasts by affinity chromatography on bonded microparticulate silicas. J Chromatogr. 1984 Aug 3;297:167–177. doi: 10.1016/s0021-9673(01)89040-3. [DOI] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Mannino R. J., Ballmer K., Zeltner D., Burger M. M. An inhibitor of animal cell growth increases cell-to-cell adhesion. J Cell Biol. 1981 Dec;91(3 Pt 1):855–859. doi: 10.1083/jcb.91.3.855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nakamura T., Nakayama Y., Ichihara A. Reciprocal modulation of growth and liver functions of mature rat hepatocytes in primary culture by an extract of hepatic plasma membranes. J Biol Chem. 1984 Jul 10;259(13):8056–8058. [PubMed] [Google Scholar]
  13. Nakamura T., Nakayama Y., Teramoto H., Nawa K., Ichihara A. Loss of reciprocal modulations of growth and liver function of hepatoma cells in culture by contact with cells or cell membranes. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6398–6402. doi: 10.1073/pnas.81.20.6398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nakamura T., Yoshimoto K., Nakayama Y., Tomita Y., Ichihara A. Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell--cell contact and cell membranes. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7229–7233. doi: 10.1073/pnas.80.23.7229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Natraj C. V., Datta P. Control of DNA synthesis in growing BALB/c 3T3 mouse cells by a fibroblast growth regulatory factor. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6115–6119. doi: 10.1073/pnas.75.12.6115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Peterson S. W., Lerch V. Inhibition of DNA synthesis in SV3T3 cultures by isolated 3T3 plasma membranes. J Cell Biol. 1983 Jul;97(1):276–279. doi: 10.1083/jcb.97.1.276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Raben D., Lieberman M. A., Glaser L. Growth inhibitory protein(s) in the 3T3 cell plasma membrane. Partial purification and dissociation of growth inhibitory events from inhibition of amino acid transport. J Cell Physiol. 1981 Jul;108(1):35–45. doi: 10.1002/jcp.1041080106. [DOI] [PubMed] [Google Scholar]
  18. Renauer D., Oesch F., Kinkel J., Unger K. K., Wieser R. J. Fractionation of membrane proteins on immobilized lectins by high-performance liquid affinity chromatography. Anal Biochem. 1985 Dec;151(2):424–427. doi: 10.1016/0003-2697(85)90198-8. [DOI] [PubMed] [Google Scholar]
  19. Rinderknecht S. B., Weiler J. M. Termination of tritiated thymidine incorporation by freezing the cells. J Immunol Methods. 1983 Dec 30;65(3):293–294. doi: 10.1016/0022-1759(83)90124-2. [DOI] [PubMed] [Google Scholar]
  20. Rohde H., Vargas L., Hahn E., Kalbfleisch H., Bruguera M., Timpl R. Radioimmunoassay for type III procollagen peptide and its application to human liver disease. Eur J Clin Invest. 1979 Dec;9(6):451–459. doi: 10.1111/j.1365-2362.1979.tb00912.x. [DOI] [PubMed] [Google Scholar]
  21. Sykes B., Puddle B., Francis M., Smith R. The estimation of two collagens from human dermis by interrupted gel electrophoresis. Biochem Biophys Res Commun. 1976 Oct 18;72(4):1472–1480. doi: 10.1016/s0006-291x(76)80180-5. [DOI] [PubMed] [Google Scholar]
  22. Whittenberger B., Glaser L. Inhibition of DNA synthesis in cultures of 3T3 cells by isolated surface membranes. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2251–2255. doi: 10.1073/pnas.74.6.2251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wieser R. J., Heck R., Oesch F. Involvement of plasma membrane glycoproteins in the contact-dependent inhibition of growth of human fibroblasts. Exp Cell Res. 1985 Jun;158(2):493–499. doi: 10.1016/0014-4827(85)90472-0. [DOI] [PubMed] [Google Scholar]
  24. Yaoi Y. Growth-inhibitory glycopeptides obtained from the cell surface of cultured chick embryo fibroblasts. Exp Cell Res. 1984 Sep;154(1):147–154. doi: 10.1016/0014-4827(84)90675-x. [DOI] [PubMed] [Google Scholar]

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