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. 1992 Jan 2;116(2):437–447. doi: 10.1083/jcb.116.2.437

Phorbol ester induced osteoclast-like differentiation of a novel human leukemic cell line (FLG 29.1)

PMCID: PMC2289288  PMID: 1309813

Abstract

Studies on human osteoclast formation have been hampered by lack of a defined isolated progenitor cell population. We describe here the establishment of a human leukemic cell line (designated FLG 29.1) from bone marrow of a patient with acute monoblastic leukemia. The cultured cells are predominantly undifferentiated leukemic blasts, but addition of 12-o-tetradecanoylphorbol 13-acetate (TPA; 0.1 microM) induces irreversible differentiation into adherent, non-dividing, multinucleated cells. TPA-treated cells bear surface antigens typical of fetal osteoclasts, degrade 45Ca-labeled devitalized bone particles, display tartrate-resistant acid phosphatase in both mononuclear and multinuclear cells and receptors for calcitonin. Calcitonin increases intracellular cAMP accumulation in TPA-treated cells. TPA-treated cells show some ultrastructural features of osteoclasts as evidenced by transmission EM. These results indicate that FLG 29.1 cells may represent an osteoclast committed cell population, which upon induction with TPA acquire some morphological, phenotypical, and functional features of differentiated osteoclasts.

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Selected References

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  1. Abe E., Miyaura C., Sakagami H., Takeda M., Konno K., Yamazaki T., Yoshiki S., Suda T. Differentiation of mouse myeloid leukemia cells induced by 1 alpha,25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4990–4994. doi: 10.1073/pnas.78.8.4990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amento E. P., Bhalla A. K., Kurnick J. T., Kradin R. L., Clemens T. L., Holick S. A., Holick M. F., Krane S. M. 1 alpha,25-dihydroxyvitamin D3 induces maturation of the human monocyte cell line U937, and, in association with a factor from human T lymphocytes, augments production of the monokine, mononuclear cell factor. J Clin Invest. 1984 Mar;73(3):731–739. doi: 10.1172/JCI111266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Athanasou N. A., Quinn J., Heryet A., McGee J. O. Localization of platelet antigens and fibrinogen on osteoclasts. J Cell Sci. 1988 Jan;89(Pt 1):115–122. doi: 10.1242/jcs.89.1.115. [DOI] [PubMed] [Google Scholar]
  4. Athanasou N. A., Quinn J., McGee J. O. Leucocyte common antigen is present on osteoclasts. J Pathol. 1987 Oct;153(2):121–126. doi: 10.1002/path.1711530205. [DOI] [PubMed] [Google Scholar]
  5. Bar-Shavit Z., Teitelbaum S. L., Reitsma P., Hall A., Pegg L. E., Trial J., Kahn A. J. Induction of monocytic differentiation and bone resorption by 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5907–5911. doi: 10.1073/pnas.80.19.5907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Barsony J., Marx S. J. Immunocytology on microwave-fixed cells reveals rapid and agonist-specific changes in subcellular accumulation patterns for cAMP or cGMP. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1188–1192. doi: 10.1073/pnas.87.3.1188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bennett J. M., Catovsky D., Daniel M. T., Flandrin G., Galton D. A., Gralnick H. R., Sultan C. Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol. 1976 Aug;33(4):451–458. doi: 10.1111/j.1365-2141.1976.tb03563.x. [DOI] [PubMed] [Google Scholar]
  8. Boyde A., Ali N. N., Jones S. J. Resorption of dentine by isolated osteoclasts in vitro. Br Dent J. 1984 Mar 24;156(6):216–220. doi: 10.1038/sj.bdj.4805313. [DOI] [PubMed] [Google Scholar]
  9. Brooker G., Harper J. F., Terasaki W. L., Moylan R. D. Radioimmunoassay of cyclic AMP and cyclic GMP. Adv Cyclic Nucleotide Res. 1979;10:1–33. [PubMed] [Google Scholar]
  10. Burger E. H., van der Meer J. W., Nijweide P. J. Osteoclast formation from mononuclear phagocytes: role of bone-forming cells. J Cell Biol. 1984 Dec;99(6):1901–1906. doi: 10.1083/jcb.99.6.1901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  12. Carbone A., Pinto A., Gloghini A., De Re V., Alosi M., Zagonel V., Tirelli U., Attadia V., Boiocchi M., Volpe R. Report of an unusual small lymphocytic B-cell lymphoma selectively involving the B-zone of lymph node. Cancer. 1990 Jul 15;66(2):302–312. doi: 10.1002/1097-0142(19900715)66:2<302::aid-cncr2820660218>3.0.co;2-q. [DOI] [PubMed] [Google Scholar]
  13. Chambers T. J., Fuller K. Bone cells predispose bone surfaces to resorption by exposure of mineral to osteoclastic contact. J Cell Sci. 1985 Jun;76:155–165. doi: 10.1242/jcs.76.1.155. [DOI] [PubMed] [Google Scholar]
  14. Chambers T. J., Horton M. A. Failure of cells of the mononuclear phagocyte series to resorb bone. Calcif Tissue Int. 1984 Sep;36(5):556–558. doi: 10.1007/BF02405365. [DOI] [PubMed] [Google Scholar]
  15. Chambers T. J., Magnus C. J. Calcitonin alters behaviour of isolated osteoclasts. J Pathol. 1982 Jan;136(1):27–39. doi: 10.1002/path.1711360104. [DOI] [PubMed] [Google Scholar]
  16. Chambers T. J., McSheehy P. M., Thomson B. M., Fuller K. The effect of calcium-regulating hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones. Endocrinology. 1985 Jan;116(1):234–239. doi: 10.1210/endo-116-1-234. [DOI] [PubMed] [Google Scholar]
  17. Dutrillaux B., Lejeune J. Sur une nouvelle technique d'analyse du caryotype humain. C R Acad Sci Hebd Seances Acad Sci D. 1971 May 17;272(20):2638–2640. [PubMed] [Google Scholar]
  18. Ejiri S. The preosteoclast and its cytodifferentiation into the osteoclast: ultrastructural and histochemical studies of rat fetal parietal bone. Arch Histol Jpn. 1983 Sep;46(4):533–557. [PubMed] [Google Scholar]
  19. Fallon M. D., Teitelbaum S. L., Kahn A. J. Multinucleation enhances macrophage-mediated bone resorption. Lab Invest. 1983 Aug;49(2):159–164. [PubMed] [Google Scholar]
  20. Fattorossi A., Nisini R., Pizzolo J. G., D'Amelio R. New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis. Cytometry. 1989 May;10(3):320–325. doi: 10.1002/cyto.990100311. [DOI] [PubMed] [Google Scholar]
  21. Hattersley G., Chambers T. J. Calcitonin receptors as markers for osteoclastic differentiation: correlation between generation of bone-resorptive cells and cells that express calcitonin receptors in mouse bone marrow cultures. Endocrinology. 1989 Sep;125(3):1606–1612. doi: 10.1210/endo-125-3-1606. [DOI] [PubMed] [Google Scholar]
  22. Hattersley G., Chambers T. J. Generation of osteoclastic function in mouse bone marrow cultures: multinuclearity and tartrate-resistant acid phosphatase are unreliable markers for osteoclastic differentiation. Endocrinology. 1989 Apr;124(4):1689–1696. doi: 10.1210/endo-124-4-1689. [DOI] [PubMed] [Google Scholar]
  23. Hefley T. J., Stern P. H. Isolation of osteoclasts from fetal rat long bones. Calcif Tissue Int. 1982 Sep;34(5):480–487. doi: 10.1007/BF02411289. [DOI] [PubMed] [Google Scholar]
  24. Hewison M., Barker S., Brennan A., Katz D. R., O'Riordan J. L. Modulation of myelomonocytic U937 cells by vitamin D metabolites. Bone Miner. 1989 Mar;5(3):323–333. doi: 10.1016/0169-6009(89)90010-x. [DOI] [PubMed] [Google Scholar]
  25. Horton M. A., Lewis D., McNulty K., Pringle J. A., Chambers T. J. Monoclonal antibodies to osteoclastomas (giant cell bone tumors): definition of osteoclast-specific cellular antigens. Cancer Res. 1985 Nov;45(11 Pt 2):5663–5669. [PubMed] [Google Scholar]
  26. Horton M. A., Rimmer E. F., Lewis D., Pringle J. A., Fuller K., Chambers T. J. Cell surface characterization of the human osteoclast: phenotypic relationship to other bone marrow-derived cell types. J Pathol. 1984 Dec;144(4):281–294. doi: 10.1002/path.1711440410. [DOI] [PubMed] [Google Scholar]
  27. Ibbotson K. J., Roodman G. D., McManus L. M., Mundy G. R. Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells. J Cell Biol. 1984 Aug;99(2):471–480. doi: 10.1083/jcb.99.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Key L. L., Jr, Ries W. L., Taylor R. G., Hays B. D., Pitzer B. L. Oxygen derived free radicals in osteoclasts: the specificity and location of the nitroblue tetrazolium reaction. Bone. 1990;11(2):115–119. doi: 10.1016/8756-3282(90)90058-7. [DOI] [PubMed] [Google Scholar]
  29. Kukita A., Bonewald L., Rosen D., Seyedin S., Mundy G. R., Roodman G. D. Osteoinductive factor inhibits formation of human osteoclast-like cells. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3023–3026. doi: 10.1073/pnas.87.8.3023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kukita T., Roodman G. D. Development of a monoclonal antibody to osteoclasts formed in vitro which recognizes mononuclear osteoclast precursors in the marrow. Endocrinology. 1989 Aug;125(2):630–637. doi: 10.1210/endo-125-2-630. [DOI] [PubMed] [Google Scholar]
  31. Kurihara N., Gluck S., Roodman G. D. Sequential expression of phenotype markers for osteoclasts during differentiation of precursors for multinucleated cells formed in long-term human marrow cultures. Endocrinology. 1990 Dec;127(6):3215–3221. doi: 10.1210/endo-127-6-3215. [DOI] [PubMed] [Google Scholar]
  32. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  33. Lindunger A., MacKay C. A., Ek-Rylander B., Andersson G., Marks S. C., Jr Histochemistry and biochemistry of tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant acid adenosine triphosphatase (TrATPase) in bone, bone marrow and spleen: implications for osteoclast ontogeny. Bone Miner. 1990 Aug;10(2):109–119. doi: 10.1016/0169-6009(90)90086-u. [DOI] [PubMed] [Google Scholar]
  34. Luk S. C., Nopajaroonsri C., Simon G. T. The ultrastructure of endosteum: a topographic study in young adult rabbits. J Ultrastruct Res. 1974 Feb;46(2):165–183. doi: 10.1016/s0022-5320(74)80054-7. [DOI] [PubMed] [Google Scholar]
  35. MacDonald B. R., Takahashi N., McManus L. M., Holahan J., Mundy G. R., Roodman G. D. Formation of multinucleated cells that respond to osteotropic hormones in long term human bone marrow cultures. Endocrinology. 1987 Jun;120(6):2326–2333. doi: 10.1210/endo-120-6-2326. [DOI] [PubMed] [Google Scholar]
  36. Malone J. D., Teitelbaum S. L., Griffin G. L., Senior R. M., Kahn A. J. Recruitment of osteoclast precursors by purified bone matrix constituents. J Cell Biol. 1982 Jan;92(1):227–230. doi: 10.1083/jcb.92.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marks S. C., Jr The origin of osteoclasts: evidence, clinical implications and investigative challenges of an extra-skeletal source. J Oral Pathol. 1983 Aug;12(4):226–256. doi: 10.1111/j.1600-0714.1983.tb00337.x. [DOI] [PubMed] [Google Scholar]
  38. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  39. Nicholson G. C., Moseley J. M., Sexton P. M., Mendelsohn F. A., Martin T. J. Abundant calcitonin receptors in isolated rat osteoclasts. Biochemical and autoradiographic characterization. J Clin Invest. 1986 Aug;78(2):355–360. doi: 10.1172/JCI112584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nicholson G. C., Moseley J. M., Yates A. J., Martin T. J. Control of cyclic adenosine 3',5'-monophosphate production in osteoclasts: calcitonin-induced persistent activation and homologous desensitization of adenylate cyclase. Endocrinology. 1987 May;120(5):1902–1908. doi: 10.1210/endo-120-5-1902. [DOI] [PubMed] [Google Scholar]
  41. Osdoby P., Martini M. C., Caplan A. I. Isolated osteoclasts and their presumed progenitor cells, the monocyte, in culture. J Exp Zool. 1982 Dec 30;224(3):331–344. doi: 10.1002/jez.1402240306. [DOI] [PubMed] [Google Scholar]
  42. Pfeilschifter J., Mundy G. R. Modulation of type beta transforming growth factor activity in bone cultures by osteotropic hormones. Proc Natl Acad Sci U S A. 1987 Apr;84(7):2024–2028. doi: 10.1073/pnas.84.7.2024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pinto A., Colletta G., Del Vecchio L., Rosati R., Attadia V., Cimino R., Colombatti A. c-fos oncogene expression in human hematopoietic malignancies is restricted to acute leukemias with monocytic phenotype and to subsets of B cell leukemias. Blood. 1987 Nov;70(5):1450–1457. [PubMed] [Google Scholar]
  44. Polack A., Hartl G., Zimber U., Freese U. K., Laux G., Takaki K., Hohn B., Gissmann L., Bornkamm G. W. A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates. Gene. 1984 Mar;27(3):279–288. doi: 10.1016/0378-1119(84)90072-6. [DOI] [PubMed] [Google Scholar]
  45. Raisz L. G., Trummel C. L., Holick M. F., DeLuca H. F. 1,25-dihydroxycholecalciferol: a potent stimulator of bone resorption in tissue culture. Science. 1972 Feb 18;175(4023):768–769. doi: 10.1126/science.175.4023.768. [DOI] [PubMed] [Google Scholar]
  46. Reinholt F. P., Hultenby K., Oldberg A., Heinegård D. Osteopontin--a possible anchor of osteoclasts to bone. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4473–4475. doi: 10.1073/pnas.87.12.4473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rodan G. A., Martin T. J. Role of osteoblasts in hormonal control of bone resorption--a hypothesis. Calcif Tissue Int. 1981;33(4):349–351. doi: 10.1007/BF02409454. [DOI] [PubMed] [Google Scholar]
  48. Roodman G. D., Ibbotson K. J., MacDonald B. R., Kuehl T. J., Mundy G. R. 1,25-Dihydroxyvitamin D3 causes formation of multinucleated cells with several osteoclast characteristics in cultures of primate marrow. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8213–8217. doi: 10.1073/pnas.82.23.8213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rovera G., Santoli D., Damsky C. Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2779–2783. doi: 10.1073/pnas.76.6.2779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rånby M., Nguyen G., Scarabin P. Y., Samama M. Immunoreactivity of tissue plasminogen activator and of its inhibitor complexes. Biochemical and multicenter validation of a two site immunosorbent assay. Thromb Haemost. 1989 Jun 30;61(3):409–414. [PubMed] [Google Scholar]
  51. Scheurich P., Ucer U., Krönke M., Pfizenmaier K. Quantification and characterization of high-affinity membrane receptors for tumor necrosis factor on human leukemic cell lines. Int J Cancer. 1986 Jul 15;38(1):127–133. doi: 10.1002/ijc.2910380120. [DOI] [PubMed] [Google Scholar]
  52. Schneider G. B., Relfson M. A bone marrow fraction enriched for granulocyte-macrophage progenitors gives rise to osteoclasts in vitro. Bone. 1988;9(5):303–308. doi: 10.1016/8756-3282(88)90014-2. [DOI] [PubMed] [Google Scholar]
  53. Seabright M. A rapid banding technique for human chromosomes. Lancet. 1971 Oct 30;2(7731):971–972. doi: 10.1016/s0140-6736(71)90287-x. [DOI] [PubMed] [Google Scholar]
  54. Sminia T., Dijkstra C. D. The origin of osteoclasts: an immunohistochemical study on macrophages and osteoclasts in embryonic rat bone. Calcif Tissue Int. 1986 Oct;39(4):263–266. doi: 10.1007/BF02555216. [DOI] [PubMed] [Google Scholar]
  55. Sutton J. S., Weiss L. Transformation of monocytes in tissue culture into macrophages, epithelioid cells, and multinucleated giant cells. An electron microscope study. J Cell Biol. 1966 Feb;28(2):303–332. doi: 10.1083/jcb.28.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Takahashi N., Akatsu T., Sasaki T., Nicholson G. C., Moseley J. M., Martin T. J., Suda T. Induction of calcitonin receptors by 1 alpha, 25-dihydroxyvitamin D3 in osteoclast-like multinucleated cells formed from mouse bone marrow cells. Endocrinology. 1988 Sep;123(3):1504–1510. doi: 10.1210/endo-123-3-1504. [DOI] [PubMed] [Google Scholar]
  57. Takahashi N., MacDonald B. R., Hon J., Winkler M. E., Derynck R., Mundy G. R., Roodman G. D. Recombinant human transforming growth factor-alpha stimulates the formation of osteoclast-like cells in long-term human marrow cultures. J Clin Invest. 1986 Oct;78(4):894–898. doi: 10.1172/JCI112677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Takahashi N., Mundy G. R., Kuehl T. J., Roodman G. D. Osteoclast-like cell formation in fetal and newborn long-term baboon marrow cultures is more sensitive to 1,25-dihydroxyvitamin D3 than adult long-term marrow cultures. J Bone Miner Res. 1987 Aug;2(4):311–317. doi: 10.1002/jbmr.5650020408. [DOI] [PubMed] [Google Scholar]
  59. Teitelbaum S. L., Stewart C. C., Kahn A. J. Rodent peritoneal macrophages as bone resorbing cells. Calcif Tissue Int. 1979 Jul 3;27(3):255–261. doi: 10.1007/BF02441194. [DOI] [PubMed] [Google Scholar]
  60. Udagawa N., Takahashi N., Akatsu T., Sasaki T., Yamaguchi A., Kodama H., Martin T. J., Suda T. The bone marrow-derived stromal cell lines MC3T3-G2/PA6 and ST2 support osteoclast-like cell differentiation in cocultures with mouse spleen cells. Endocrinology. 1989 Oct;125(4):1805–1813. doi: 10.1210/endo-125-4-1805. [DOI] [PubMed] [Google Scholar]
  61. Udagawa N., Takahashi N., Akatsu T., Tanaka H., Sasaki T., Nishihara T., Koga T., Martin T. J., Suda T. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7260–7264. doi: 10.1073/pnas.87.18.7260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. de Vernejoul M. C., Horowitz M., Demignon J., Neff L., Baron R. Bone resorption by isolated chick osteoclasts in culture is stimulated by murine spleen cell supernatant fluids (osteoclast-activating factor) and inhibited by calcitonin and prostaglandin E2. J Bone Miner Res. 1988 Feb;3(1):69–80. doi: 10.1002/jbmr.5650030111. [DOI] [PubMed] [Google Scholar]

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