Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Apr 1;104(4):1105–1115. doi: 10.1083/jcb.104.4.1105

Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction

PMCID: PMC2114448  PMID: 3104350

Abstract

To establish the relation of glycosaminoglycan synthesis to cell proliferation, we investigated the synthesis of individual glycosaminoglycan species by intact cells and in a cell-free system, using normal and transformed human fibroblasts under differing culture conditions. Reducing serum concentration brought about a marked decline in the synthesis of hyaluronate (HA) as well as cell proliferation on both normal and transformed cells. Both HA synthesis and proliferation decreased with increasing cell densities markedly (in inverse proportion to cell density) in normal cells but gradually in transformed cells. This noticeable congruity of the changes in HA synthesis and proliferation indicates that the change in HA synthesis is related primarily to cell proliferation rather than to cell density or cellular transformation. Examination of HA synthesis in a cell-free system demonstrated that the activity of HA synthetase also fluctuated in conjunction with cell proliferation. Furthermore, growth-reduced cells (except crowded transformed cells) inhibited cell-free HA synthesis and this inhibition was induced coincidentally with a decrease in both HA synthetase activity and proliferation. These findings suggest that the change in HA synthesis is significant in the regulation of cell proliferation.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BITTER T., MUIR H. M. A modified uronic acid carbazole reaction. Anal Biochem. 1962 Oct;4:330–334. doi: 10.1016/0003-2697(62)90095-7. [DOI] [PubMed] [Google Scholar]
  2. Bartold P. M., Boyd R. R., Page R. C. Proteoglycans synthesized by gingival fibroblasts derived from human donors of different ages. J Cell Physiol. 1986 Jan;126(1):37–46. doi: 10.1002/jcp.1041260106. [DOI] [PubMed] [Google Scholar]
  3. Bhavanandan V. P., Davidson E. A. Mucopolysaccharides associated with nuclei of cultured mammalian cells. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2032–2036. doi: 10.1073/pnas.72.6.2032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bischoff E., Tran-Thi T. A., Decker K. F. Nucleotide pyrophosphatase of rat liver. A comparative study on the enzymes solubilized and purified from plasma membrane and endoplasmic reticulum. Eur J Biochem. 1975 Feb 21;51(2):353–361. doi: 10.1111/j.1432-1033.1975.tb03935.x. [DOI] [PubMed] [Google Scholar]
  5. Busa W. B., Nuccitelli R. Metabolic regulation via intracellular pH. Am J Physiol. 1984 Apr;246(4 Pt 2):R409–R438. doi: 10.1152/ajpregu.1984.246.4.R409. [DOI] [PubMed] [Google Scholar]
  6. Castor L. N. Contact inhibitions of cell division and cell movement. J Invest Dermatol. 1972 Jul;59(1):27–32. doi: 10.1111/1523-1747.ep12625732. [DOI] [PubMed] [Google Scholar]
  7. Cohn R. H., Cassiman J. J., Bernfield M. R. Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan. J Cell Biol. 1976 Oct;71(1):280–294. doi: 10.1083/jcb.71.1.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans W. H., Gurd J. W. Properties of a 5'-nucleotidase purified from mouse liver plasma membranes. Biochem J. 1973 May;133(1):189–199. doi: 10.1042/bj1330189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fedarko N. S., Conrad H. E. A unique heparan sulfate in the nuclei of hepatocytes: structural changes with the growth state of the cells. J Cell Biol. 1986 Feb;102(2):587–599. doi: 10.1083/jcb.102.2.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Furukawa K., Bhavanandan V. P. Influence of glycosaminoglycans on endogenous DNA synthesis in isolated normal and cancer cell nuclei. Differential effect of heparin. Biochim Biophys Acta. 1982 Jun 30;697(3):344–352. doi: 10.1016/0167-4781(82)90098-7. [DOI] [PubMed] [Google Scholar]
  12. Furukawa K., Bhavanandan V. P. Influences of anionic polysaccharides on DNA synthesis in isolated nuclei and by DNA polymerase alpha: correlation of observed effects with properties of the polysaccharides. Biochim Biophys Acta. 1983 Sep 9;740(4):466–475. doi: 10.1016/0167-4781(83)90096-9. [DOI] [PubMed] [Google Scholar]
  13. Furukawa K., Terayama H. Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver. Biochim Biophys Acta. 1977 Sep 29;499(2):278–289. doi: 10.1016/0304-4165(77)90010-1. [DOI] [PubMed] [Google Scholar]
  14. Gallagher J. T., Lyon M., Steward W. P. Structure and function of heparan sulphate proteoglycans. Biochem J. 1986 Jun 1;236(2):313–325. doi: 10.1042/bj2360313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gebb C., Baron D., Grisebach H. Spectroscopic evidence for the formation of a 4-keto intermediate in the UDP-apiose/UDP-xylose synthase reaction. Eur J Biochem. 1975 Jun;54(2):493–498. doi: 10.1111/j.1432-1033.1975.tb04161.x. [DOI] [PubMed] [Google Scholar]
  16. Hata R., Nagai Y. A micro colorimetric determination of acidic glycosaminoglycans by two dimensional electrophoresis on a cellulose acetate strip. Anal Biochem. 1973 Apr;52(2):652–656. doi: 10.1016/0003-2697(73)90075-4. [DOI] [PubMed] [Google Scholar]
  17. Honda A., Iwama M., Umeda T., Mori Y. The teratogenic mechanism of 6-aminonicotinamide on limb formation of chick embryos: abnormalities in the biosynthesis of glycosaminoglycans and proteoglycans in micromelia. J Biochem. 1982 Jun;91(6):1959–1970. doi: 10.1093/oxfordjournals.jbchem.a133890. [DOI] [PubMed] [Google Scholar]
  18. Hopwood J. J., Dorfman A. Glycosaminoglycan synthesis by cultured human skin fibroblasts after transformation with simian virus 40. J Biol Chem. 1977 Jul 25;252(14):4777–4785. [PubMed] [Google Scholar]
  19. Hovingh P., Linker A. The enzymatic degradation of heparin and heparitin sulfate. 3. Purification of a heparitinase and a heparinase from flavobacteria. J Biol Chem. 1970 Nov 25;245(22):6170–6175. [PubMed] [Google Scholar]
  20. Iozzo R. V. Proteoglycans: structure, function, and role in neoplasia. Lab Invest. 1985 Oct;53(4):373–396. [PubMed] [Google Scholar]
  21. Kawakami H., Terayama H. Liver plasma membranes and proteoglycan prepared therefrom inhibit the growth of hepatoma cells in vitro. Biochim Biophys Acta. 1981 Aug 6;646(1):161–168. doi: 10.1016/0005-2736(81)90283-2. [DOI] [PubMed] [Google Scholar]
  22. Kim J. J., Conrad H. E. Kinetics of mucopolysaccharide and glycoprotein synthesis by chick embryo chondrocytes. Effect of D-glucose concentration in the culture medium. J Biol Chem. 1976 Oct 25;251(20):6210–6217. [PubMed] [Google Scholar]
  23. Kraemer P. M., Tobey R. A. Cell-cycle dependent desquamation of heparan sulfate from the cell surface. J Cell Biol. 1972 Dec;55(3):713–717. doi: 10.1083/jcb.55.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lembach K. J. Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblasts by mouse epidermal growth factor. J Cell Physiol. 1976 Oct;89(2):277–288. doi: 10.1002/jcp.1040890211. [DOI] [PubMed] [Google Scholar]
  25. Margolis R. K., Crockett C. P., Kiang W. L., Margolis R. U. Glycosaminoglycans and glycoproteins associated with rat brain nuclei. Biochim Biophys Acta. 1976 Dec 21;451(2):465–469. doi: 10.1016/0304-4165(76)90141-0. [DOI] [PubMed] [Google Scholar]
  26. Marin G., Wynford-Thomas D., Lamontagne A., Prescott D. M. Density-dependent regulation of growth in somatic hybrids between normal Chinese hamster fibroblasts and V79-8 (G1-) cells. Exp Cell Res. 1984 Dec;155(2):575–582. doi: 10.1016/0014-4827(84)90217-9. [DOI] [PubMed] [Google Scholar]
  27. Matuoka K., Mitsui Y. Changes in cell-surface glycosaminoglycans in human diploid fibroblasts during in vitro aging. Mech Ageing Dev. 1981 Feb;15(2):153–163. doi: 10.1016/0047-6374(81)90071-3. [DOI] [PubMed] [Google Scholar]
  28. Matuoka K., Mitsui Y., Murota S. Growth-coupled changes in glucosaminoglycans (heparan sulfate and hyaluronic acid) in normal and transformed human fibroblasts. Cell Biol Int Rep. 1985 Jun;9(6):577–586. doi: 10.1016/0309-1651(85)90023-2. [DOI] [PubMed] [Google Scholar]
  29. Matuoka K., Mitsui Y., Murota S. Heparan sulfate enhances growth of transformed human cells. Cell Struct Funct. 1984 Dec;9(4):357–367. doi: 10.1247/csf.9.357. [DOI] [PubMed] [Google Scholar]
  30. Matuoka K., Mitsui Y., Murota S., Namba M. Actions of exogenous heparan sulfate and hyaluronic acid on growth and thymidine incorporation of normal and transformed human fibroblasts. A comparison with the effects of high cell density and low serum concentration and a warning against thymidine incorporation as a measure of DNA synthesis. Cell Biol Int Rep. 1985 Sep;9(9):815–824. doi: 10.1016/0309-1651(85)90100-6. [DOI] [PubMed] [Google Scholar]
  31. Mian N. Analysis of cell-growth-phase-related variations in hyaluronate synthase activity of isolated plasma-membrane fractions of cultured human skin fibroblasts. Biochem J. 1986 Jul 15;237(2):333–342. doi: 10.1042/bj2370333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mian N. Characterization of a high-Mr plasma-membrane-bound protein and assessment of its role as a constituent of hyaluronate synthase complex. Biochem J. 1986 Jul 15;237(2):343–357. doi: 10.1042/bj2370343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moolenaar W. H., Tertoolen L. G., de Laat S. W. Phorbol ester and diacylglycerol mimic growth factors in raising cytoplasmic pH. Nature. 1984 Nov 22;312(5992):371–374. doi: 10.1038/312371a0. [DOI] [PubMed] [Google Scholar]
  34. Moscatelli D., Rubin H. Hormonal control of hyaluronic acid production in fibroblasts and its relation to nucleic acid and protein synthesis. J Cell Physiol. 1977 Apr;91(1):79–88. doi: 10.1002/jcp.1040910109. [DOI] [PubMed] [Google Scholar]
  35. Murota S., Abe M., Otsuka K. Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (3) induction of hyaluronic acid synthetase by prostaglandin F2alpha. Prostaglandins. 1977 Nov;14(5):983–991. doi: 10.1016/0090-6980(77)90313-6. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Namba M., Kusaka T., Fukushima F., Kimoto T. Differential growth response of normal human diploid fibroblasts and in vitro transformed human fibroblasts in serum-free defined culture medium. Int J Cancer. 1984 Nov 15;34(5):645–649. doi: 10.1002/ijc.2910340510. [DOI] [PubMed] [Google Scholar]
  38. Namba M., Nishitani K., Kimoto T. Characteristics of WI-38 cells (WI-38 CT-1) transformed by treatment with Co-60 gamma rays. Gan. 1980 Jun;71(3):300–307. [PubMed] [Google Scholar]
  39. Ohya T., Kaneko Y. Novel hyaluronidase from streptomyces. Biochim Biophys Acta. 1970 Mar 18;198(3):607–609. doi: 10.1016/0005-2744(70)90139-7. [DOI] [PubMed] [Google Scholar]
  40. Philipson L. H., Schwartz N. B. Subcellular localization of hyaluronate synthetase in oligodendroglioma cells. J Biol Chem. 1984 Apr 25;259(8):5017–5023. [PubMed] [Google Scholar]
  41. Piepkorn M., Carney H., Linker A. Growth-related variations in the glycosaminoglycan synthesis of ultraviolet light-induced murine cutaneous fibrosarcoma cells. Lab Invest. 1985 Aug;53(2):187–193. [PubMed] [Google Scholar]
  42. Poole A. R. Proteoglycans in health and disease: structures and functions. Biochem J. 1986 May 15;236(1):1–14. doi: 10.1042/bj2360001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Prehm P. Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth. Biochem J. 1983 Apr 1;211(1):191–198. doi: 10.1042/bj2110191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Riesenfeld J., Hözok M., Lindahl U. Biosynthesis of heparan sulfate in rat liver. Characterization of polysaccharides obtained with intact cells and with a cell-free system. J Biol Chem. 1982 Jun 25;257(12):7050–7055. [PubMed] [Google Scholar]
  45. Rodén L., Koerner T., Olson C., Schwartz N. B. Mechanisms of chain initiation in the biosynthesis of connective tissue polysaccharides. Fed Proc. 1985 Feb;44(2):373–380. [PubMed] [Google Scholar]
  46. Sisson J. C., Castor C. W., Klavons J. A. Connective tissue activation. XVIII. Stimulation of hyaluronic acid synthetase activity. J Lab Clin Med. 1980 Aug;96(2):189–197. [PubMed] [Google Scholar]
  47. Sluke G., Schachtschabel D. O., Wever J. Age-related changes in the distribution pattern of glycosaminoglycans synthesized by cultured human diploid fibroblasts (WI-38). Mech Ageing Dev. 1981 May;16(1):19–27. doi: 10.1016/0047-6374(81)90028-2. [DOI] [PubMed] [Google Scholar]
  48. Smith J. A., Martin L. Do cells cycle? Proc Natl Acad Sci U S A. 1973 Apr;70(4):1263–1267. doi: 10.1073/pnas.70.4.1263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stein G. S., Roberts R. M., Davis J. L., Head W. J., Stein J. L., Thrall C. L., Van Veen J., Welch D. W. Are glycoproteins and glycosaminoglycans components of the eukaryotic genome? Nature. 1975 Dec 18;258(5536):639–641. doi: 10.1038/258639a0. [DOI] [PubMed] [Google Scholar]
  50. Tomida M., Koyama H., Ono T. Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures. J Cell Physiol. 1975 Aug;86(1):121–130. doi: 10.1002/jcp.1040860114. [DOI] [PubMed] [Google Scholar]
  51. Ullrich S. J., Hawkes S. P. The effect of the tumor promoter, phorbol myristate acetate (PMA), on hyaluronic acid (HA) synthesis by chicken embryo fibroblasts. Exp Cell Res. 1983 Oct 15;148(2):377–386. doi: 10.1016/0014-4827(83)90160-x. [DOI] [PubMed] [Google Scholar]
  52. Wever J., Schachtschabel D. O., Sluke G., Wever G. Effect of short- or long-term treatment with exogenous glycosaminoglycans on growth and glycosaminoglycan synthesis of human fibroblasts (WI-38) in culture. Mech Ageing Dev. 1980 Sep-Oct;14(1-2):89–99. doi: 10.1016/0047-6374(80)90108-6. [DOI] [PubMed] [Google Scholar]
  53. Winterbourne D. J., Salisbury J. G. Heparan sulphate is a potent inhibitor of DNA synthesis in vitro. Biochem Biophys Res Commun. 1981 Jul 16;101(1):30–37. doi: 10.1016/s0006-291x(81)80006-x. [DOI] [PubMed] [Google Scholar]
  54. Yamagata T., Saito H., Habuchi O., Suzuki S. Purification and properties of bacterial chondroitinases and chondrosulfatases. J Biol Chem. 1968 Apr 10;243(7):1523–1535. [PubMed] [Google Scholar]
  55. Yurchenco P. D., Ceccarini C., Atkinson P. H. Labeling complex carbohydrates of animal cells with monosaccharides. Methods Enzymol. 1978;50:175–204. doi: 10.1016/0076-6879(78)50019-0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES