Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1978 Apr 15;172(1):97–107. doi: 10.1042/bj1720097

The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture.

D K Fast, R Felix, C Dowse, W F Neuman, H Fleisch
PMCID: PMC1185667  PMID: 656078

Abstract

1. The effects of two diphosphonates (compounds containing a P-C-P bond), disodium dichloromethanediphosphonate and disodium 1-hydroxyethane-1,1-diphosphonate, on the metabolism of cultured rat calvaria cells, rabbit ear cartilage cells and rat skin fibroblasts were investigated. 2. The diphosphonates had no effect on the growth of cartilage cells and on the exponential growth of the calvaria cells and the fibroblasts. However, dichloromethanediphosphonate stopped the growth of the calvaria cells and the fibroblasts after the beginning of confluence, whereas the untreated cells were still growing to a certain extent. This inhibition was dose-dependent. After the drug was withdrawn, the cells recovered slowly. 1-Hydroxyethane-1,1-diphosphonate had no detectable effect on the growth of any of the cell types studied. Both diphosphonates decreased the cloning efficiency of calvaria cells and fibroblasts. 3. The K+ content of cartilage, calvaria and skin cells was diminished only by the highest (0.25 mM) concentration of dichloromethanediphosphonate. 4. Radioactive dichloromethanediphosphonate and 1-hydroxyethane-1,1-diphosphonate were taken up linearly with time for at least 48 h by calvaria cells and fibroblasts. The diphosphonate concentration in the cells depended on its concentration in the medium. 5. Both diphosphonates, in a dose-dependent fashion, markedly inhibited glycolysis, dichloromethanediphosphonate being more effective than 1-hydroxyethane-1,1-diphosphonate, at drug doses that had no effect on cell growth or cellular K+ content. Calvaria cells were much more sensitive than cartilage cells. When cartilage cells were cultured in an N2 atmosphere, these effects on glucose and lactate metabolism disappeared. 6. As increased acid production appears to be associated with resorption of bone, this decrease in lactate may explain why diphosphonates are effective inhibitors of bone resorption in vivo.

Full text

PDF
99

Selected References

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

  1. Altman R. D., Johnston C. C., Khairi M. R., Wellman H., Serafini A. N., Sankey R. R. Influence of disodium etidronate on clinical and laboratory manifestations of Paget's disease of bone (osteitis deformans). N Engl J Med. 1973 Dec 27;289(26):1379–1384. doi: 10.1056/NEJM197312272892601. [DOI] [PubMed] [Google Scholar]
  2. BORLE A. B., NICHOLS N., NICHOLS G., Jr Metabolic studies of bone in vitro. I. Normal bone. J Biol Chem. 1960 Apr;235:1206–1210. [PubMed] [Google Scholar]
  3. BURTON K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J. 1956 Feb;62(2):315–323. doi: 10.1042/bj0620315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. EAGLE H. Amino acid metabolism in mammalian cell cultures. Science. 1959 Aug 21;130(3373):432–437. doi: 10.1126/science.130.3373.432. [DOI] [PubMed] [Google Scholar]
  5. Fleisch H. A., Russell R. G., Bisaz S., Mühlbauer R. C., Williams D. A. The inhibitory effect of phosphonates on the formation of calcium phosphate crystals in vitro and on aortic and kidney calcification in vivo. Eur J Clin Invest. 1970 Mar;1(1):12–18. doi: 10.1111/j.1365-2362.1970.tb00591.x. [DOI] [PubMed] [Google Scholar]
  6. Fleisch H., Russell R. G., Francis M. D. Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture and in vivo. Science. 1969 Sep 19;165(3899):1262–1264. doi: 10.1126/science.165.3899.1262. [DOI] [PubMed] [Google Scholar]
  7. Francis M. D., Russell R. G., Fleisch H. Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo. Science. 1969 Sep 19;165(3899):1264–1266. doi: 10.1126/science.165.3899.1264. [DOI] [PubMed] [Google Scholar]
  8. Gasser A. B., Morgan D. B., Fleisch H. A., Richelle L. J. The influence of two diphosphonates on calcium metabolism in the rat. Clin Sci. 1972 Jul;43(1):31–45. doi: 10.1042/cs0430031. [DOI] [PubMed] [Google Scholar]
  9. Guilland D. F., Fleisch H. The effect of in vivo treatment with EHDP and/or 1,25-DHCC on calcium uptake and release in isolated kidney mitochondria. Biochem Biophys Res Commun. 1974 Dec 11;61(3):906–911. doi: 10.1016/0006-291x(74)90241-1. [DOI] [PubMed] [Google Scholar]
  10. Guilland D. F., Sallis J. D., Fleisch H. The effect of two diphosphonates on the handling of calcium by rat kidney mitochondria in vitro. Calcif Tissue Res. 1974;15(4):303–314. doi: 10.1007/BF02059065. [DOI] [PubMed] [Google Scholar]
  11. Guncaga J., Lauffenburger T., Lentner C., Dambacher M. A., Haas H. G., Fleisch H., Olah A. J. Diphosphonate treatment of Paget's disease of bone. A correlated metabolic, calcium kinetic and morphometric study. Horm Metab Res. 1974 Jan;6(1):62–69. [PubMed] [Google Scholar]
  12. Jung A., Bisaz S., Fleisch H. The binding of pyrophosphate and two diphosphonates by hydroxyapatite crystals. Calcif Tissue Res. 1973 Mar 30;11(4):269–280. doi: 10.1007/BF02547227. [DOI] [PubMed] [Google Scholar]
  13. Leyva A., Jr, Kelley W. N. Measurement of DNA in cultured human cells. Anal Biochem. 1974 Nov;62(1):173–179. doi: 10.1016/0003-2697(74)90378-9. [DOI] [PubMed] [Google Scholar]
  14. Morgan D. B., Monod A., Russel R. G., Fleisch H. Influence of dichloromethylene diphosphonate (Cl2MDP) and calcitonin on bone resorption, lactate production and phosphatase and pyrophosphatase content of mouse calvaria treated with parathyroid hormone in vitro. Calcif Tissue Res. 1973 Dec 31;13(4):287–294. doi: 10.1007/BF02015418. [DOI] [PubMed] [Google Scholar]
  15. Nolen G. A., Buehler E. V. The effects of disodium etidronate on the reproductive functions and embryogeny of albino rats and New Zealand rabbits. Toxicol Appl Pharmacol. 1971 Mar;18(3):548–561. doi: 10.1016/s0041-008x(71)80008-x. [DOI] [PubMed] [Google Scholar]
  16. PECK W. A., BIRGE S. J., Jr, FEDAK S. A. BONE CELLS: BIOCHEMICAL AND BIOLOGICAL STUDIES AFTER ENZYMATIC ISOLATION. Science. 1964 Dec 11;146(3650):1476–1477. doi: 10.1126/science.146.3650.1476. [DOI] [PubMed] [Google Scholar]
  17. Reynolds J. J., Minkin C., Morgan D. B., Spycher D., Fleisch H. The effect of two diphosphonates on the resorption of mouse calvaria in vitro. Calcif Tissue Res. 1972;10(4):302–313. doi: 10.1007/BF02012561. [DOI] [PubMed] [Google Scholar]
  18. Russell R. G., Kislig A. M., Casey P. A., Fleisch H., Thornton J., Schenk R., Williams D. A. Effect of diphosphonates and calcitonin on the chemistry and quantitative histology of rat bone. Calcif Tissue Res. 1973 Mar 12;11(3):179–195. doi: 10.1007/BF02547218. [DOI] [PubMed] [Google Scholar]
  19. Russell R. G., Mühlbauer R. C., Bisaz S., Williams D. A., Fleisch H. The influence of pyrophosphate, condensed phosphates, phosphonates and other phosphate compounds on the dissolution of hydroxyapatite in vitro and on bone resorption induced by parathyroid hormone in tissue culture and in thyroparathyroidectomised rats. Calcif Tissue Res. 1970;6(3):183–196. doi: 10.1007/BF02196199. [DOI] [PubMed] [Google Scholar]
  20. Russell R. G., Smith R., Preston C., Walton R. J., Woods C. G. Diphosphonates in Paget's disease. Lancet. 1974 May 11;1(7863):894–898. doi: 10.1016/s0140-6736(74)90347-x. [DOI] [PubMed] [Google Scholar]
  21. Schenk R., Merz W. A., Mühlbauer R., Russell R. G., Fleisch H. Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl 2 MDP) on the calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif Tissue Res. 1973 Mar 12;11(3):196–214. doi: 10.1007/BF02547219. [DOI] [PubMed] [Google Scholar]
  22. Troehler U., Bonjour J. P., Fleisch H. Renal secretion of diphosphonates in rats. Kidney Int. 1975 Jul;8(1):6–13. doi: 10.1038/ki.1975.70. [DOI] [PubMed] [Google Scholar]
  23. Vaes G. On the mechanisms of bone resorption. The action of parathyroid hormone on the excretion and synthesis of lysosomal enzymes and on the extracellular release of acid by bone cells. J Cell Biol. 1968 Dec;39(3):676–697. doi: 10.1083/jcb.39.3.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Walton R. J., Russell R. G., Smith R. Changes in the renal and extrarenal handling of phosphate induced by disodium etidronate (EHDP) in man. Clin Sci Mol Med. 1975 Jul;49(1):45–56. doi: 10.1042/cs0490045. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES