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. 1978 Dec 15;176(3):695–704. doi: 10.1042/bj1760695

Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

Ada G Milenkovic *, Martin Rachmeler †, Terry C Johnson *
PMCID: PMC1186291  PMID: 218551

Abstract

The presence of 1.0mm-dibutyryl cyclic AMP (N6,O2′-dibutyryladenosine 3′:5′-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24–36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [3H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [3H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [14C]fucose and [3H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse–chased with [14C]fucose and [3H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins.

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Selected References

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  1. Atkinson P. H. Synthesis and assembly of HeLa cell plasma membrane glycoproteins and proteins. J Biol Chem. 1975 Mar 25;250(6):2123–2134. [PubMed] [Google Scholar]
  2. Barbarese E., Sauerwein H., Simpkins H. Alterations in the surface glycoproteins of chicken erythrocytes following transformation with erythroblastosis strain R virus. J Membr Biol. 1973;13(2):129–142. doi: 10.1007/BF01868224. [DOI] [PubMed] [Google Scholar]
  3. Bennett G., Leblond C. P. Formation of cell coat material for the whole surface of columnar cells in the rat small intestine, as visualized by radioautography with L-fucose-3H. J Cell Biol. 1970 Aug;46(2):409–416. doi: 10.1083/jcb.46.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bennett G., Leblond C. P., Haddad A. Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radioautography after labelled fucose injection into rats. J Cell Biol. 1974 Jan;60(1):258–284. doi: 10.1083/jcb.60.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bosmann H. B., Winston R. A. Synthesis of glycoprotein, glycolipid, protein, and lipid in synchronized L5178Y cells. J Cell Biol. 1970 Apr;45(1):23–33. doi: 10.1083/jcb.45.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burger M. M. Proteolytic enzymes initiating cell division and escape from contact inhibition of growth. Nature. 1970 Jul 11;227(5254):170–171. doi: 10.1038/227170a0. [DOI] [PubMed] [Google Scholar]
  7. Chou I. N., Black P. H., Roblin R. O. Non-selective inhibition of transformed cell growth by a protease inhibitor. Proc Natl Acad Sci U S A. 1974 May;71(5):1748–1752. doi: 10.1073/pnas.71.5.1748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Collard J. G., Smets L. A. Effect of proteolytic inhibitors on growth and surface architecture of normal and transformed cells. Exp Cell Res. 1974 May;86(1):75–80. doi: 10.1016/0014-4827(74)90650-8. [DOI] [PubMed] [Google Scholar]
  9. Dehlinger P. J., Schimke R. T. Size distribution of membrane proteins of rat liver and their relative rates of degradation. J Biol Chem. 1971 Apr 25;246(8):2574–2583. [PubMed] [Google Scholar]
  10. Frank W. Cyclic 3':5' AMP and cell proliferation in cultures of embryonic rat cells. Exp Cell Res. 1972 Mar;71(1):238–241. doi: 10.1016/0014-4827(72)90287-x. [DOI] [PubMed] [Google Scholar]
  11. Froehlich J. E., Rachmeler M. Effect of adenosine 3'-5'-cyclic monophosphate on cell proliferation. J Cell Biol. 1972 Oct;55(1):19–31. doi: 10.1083/jcb.55.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gahmberg C. G., Hakomori S. Organization of glycolipids and glycoproteins in surface membranes: dependency on cell cycle and on transformation. Biochem Biophys Res Commun. 1974 Jul 10;59(1):283–291. doi: 10.1016/s0006-291x(74)80204-4. [DOI] [PubMed] [Google Scholar]
  13. Gerner E. W., Glick M. C., Warren L. Membranes of animal cells. V. Biosynthesis of the surface membrane during the cell cycle. J Cell Physiol. 1970 Jun;75(3):275–279. doi: 10.1002/jcp.1040750303. [DOI] [PubMed] [Google Scholar]
  14. Glick M. C., Rabinowitz Z., Sachs L. Surface membrane glycopeptides which coincide with virus transformation and tumorigenesis. J Virol. 1974 May;13(5):967–974. doi: 10.1128/jvi.13.5.967-974.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Graham J. M., Sumner M. C., Curtis D. H., Pasternak C. A. Sequence of events in plasma membrane assembly during the cell cycle. Nature. 1973 Nov 30;246(5431):291–295. doi: 10.1038/246291a0. [DOI] [PubMed] [Google Scholar]
  16. Greengard P. Phosphorylated proteins as physiological effectors. Science. 1978 Jan 13;199(4325):146–152. doi: 10.1126/science.22932. [DOI] [PubMed] [Google Scholar]
  17. Hudson J. E., Johnson T. C. Rapidly metabolized glycoproteins in a neuroblastoma cell line. Biochim Biophys Acta. 1977 Apr 27;497(2):567–577. doi: 10.1016/0304-4165(77)90213-6. [DOI] [PubMed] [Google Scholar]
  18. Hudson J. E., Johnson T. C. The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line. Biochem J. 1977 Aug 15;166(2):217–223. doi: 10.1042/bj1660217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hynes R. O., Bye J. M. Density and cell cycle dependence of cell surface proteins in hamster fibroblasts. Cell. 1974 Oct;3(2):113–120. doi: 10.1016/0092-8674(74)90114-7. [DOI] [PubMed] [Google Scholar]
  20. Kaplan J., Moskowitz M. Studies on the turnover of plasma membranes in cultured mammalian cells. II. Demonstration of heterogeneous rates of turnover for plasma membrane proteins and glycoproteins. Biochim Biophys Acta. 1975 May 6;389(2):306–313. doi: 10.1016/0005-2736(75)90323-5. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Landry J., Marceau N. The relative rates of degradation of the plasma membrane glycoproteins from normal rat liver. Biochim Biophys Acta. 1975 Apr 21;389(1):154–161. doi: 10.1016/0005-2736(75)90393-4. [DOI] [PubMed] [Google Scholar]
  23. Mathews R. A., Johnson T. C., Hudson J. E. Synthesis and turnover of plasma-membrane proteins and glycoproteins in a neuroblastoma cell line. Biochem J. 1976 Jan 15;154(1):57–64. doi: 10.1042/bj1540057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nowakowski M., Atkinson P. H., Summers D. F. Incorporation of fucose into HeLa cell plasma membranes during the cell cycle. Biochim Biophys Acta. 1972 Apr 14;266(1):154–160. doi: 10.1016/0005-2736(72)90131-9. [DOI] [PubMed] [Google Scholar]
  25. Ozanne B., Sambrook J. Binding of radioactively labelled concanavalin A and wheat germ agglutinin to normal and virus-transformed cells. Nat New Biol. 1971 Aug 4;232(31):156–160. doi: 10.1038/newbio232156a0. [DOI] [PubMed] [Google Scholar]
  26. Prasad K. N., Kumar S., Gilmer K., Vernadakis A. Cyclic AMP-induced differentiated neuroblastoma cells: changes in total nucleic acid and protein contents. Biochem Biophys Res Commun. 1973 Feb 20;50(4):973–977. doi: 10.1016/0006-291x(73)91501-5. [DOI] [PubMed] [Google Scholar]
  27. Prasad K. N., Sheppard J. R. Neuroblastoma cell culture: membrane changes during cyclic AMP-induced morphological differentiation. Proc Soc Exp Biol Med. 1972 Oct;141(1):240–243. doi: 10.3181/00379727-141-36750. [DOI] [PubMed] [Google Scholar]
  28. Rieber M., Bacalao J., Alonso G. Turnover of high-molecular-weight cell surface proteins during growth and expression of malignant transformation. Cancer Res. 1975 Aug;35(8):2104–2108. [PubMed] [Google Scholar]
  29. Sheppard J. R. Restoration of contact-inhibited growth to transformed cells by dibutyryl adenosine 3':5'-cyclic monophosphate. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1316–1320. doi: 10.1073/pnas.68.6.1316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shin B. C., Ebner K. E., Hudson B. G., Carraway K. L. Membrane glycoprotein differences between normal lactating mammary tissue and the R3230 AC mammary tumor. Cancer Res. 1975 May;35(5):1135–1140. [PubMed] [Google Scholar]
  31. Shoham J., Sachs L. Different cyclic changes in the surface membrane of normal and malignant transformed cells. Exp Cell Res. 1974 Mar 30;85(1):8–14. doi: 10.1016/0014-4827(74)90206-7. [DOI] [PubMed] [Google Scholar]
  32. Truding R., Shelanski M. L., Morell P. Glycoproteins released into the culture medium of differentiating murine neuroblastoma cells. J Biol Chem. 1975 Dec 25;250(24):9348–9354. [PubMed] [Google Scholar]
  33. Tweto J., Doyle D. Turnover of the plasma membrane proteins of hepatoma tissue culture cells. J Biol Chem. 1976 Feb 10;251(3):872–882. [PubMed] [Google Scholar]
  34. Warren L., Fuhrer J. P., Buck C. A. Surface glycoproteins of cells before and after transformation by oncogenic viruses. Fed Proc. 1973 Jan;32(1):80–85. [PubMed] [Google Scholar]

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