Methods for studying cell death in bone

  • Brendan F. Boyce
  • David E. Hughes
  • Kenneth R. Wright


Apoptosis is a common form of cell death that controls cell numbers in most tissues during their development as well as in a wide variety of normal and pathological settings. It has been defined by a series of characteristic morphological changes that take place in nuclear chromatin and cytoplasm of cells dying by a mechanism that differs from ischaemic necrosis in that it typically affects single cells, rather than groups of cells [1, 2], These changes appear to be regulated in affected cells; hence the alternative terms: programmed, biological, physiological or controlled cell death.


Apoptotic Cell Apoptotic Body Mineral Research Nuclear Chromatin Hypertrophic Chondrocytes 


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  1. 1.
    Kerr, J.F.R., Wyllie, A.H. and Currie, A.R. (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26), 239–257.PubMedGoogle Scholar
  2. 2.
    Wyllie, A.H., Kerr, J.F.R. and Currie, A.R. (1980) Cell death: the significance of apoptosis. International Reviews of Cytology 68), 251–306.Google Scholar
  3. 3.
    Zou, H. and Niswander, L. (1996) Requirement for BMP signaling in interdigital apoptosis and scale formation. Science 272), 738–741.PubMedCrossRefGoogle Scholar
  4. 4.
    Mooney, E.E., Ruis Peris, J.M., O’Neill, A. and Sweeney, E.C. (1995) Apoptotic and mitotic indices in malignant melanoma and basal cell carcinoma. Journal of Clinical Pathology 48, 242–244.PubMedGoogle Scholar
  5. 5.
    Lee, J.M. and Bernstein, A. (1993) p53 Mutations increase resistance to ionizing radiation. Proceedings of the National Academy of Sciences USA 90, 5742–5746.CrossRefGoogle Scholar
  6. 6.
    Lowe, S.W., Ruley, H.E., Jacks, T. and Housman, D.E. (1993) p53-Dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74, 957–967.PubMedCrossRefGoogle Scholar
  7. 7.
    Reed, J.C. (1994) Bcl-2 and the regulation of programmed cell death. Journal of Cell Biology 124), 1–6.PubMedCrossRefGoogle Scholar
  8. 8.
    Levine, B., Huang, Q., Isaacs, J.T. et al (1993) Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene. Nature 361), 739–742.PubMedCrossRefGoogle Scholar
  9. 9.
    Boyd, J.M., Malstrom, S., Subramanian, T. et al. (1994) Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell 79), 341–351.PubMedCrossRefGoogle Scholar
  10. 10.
    Henderson, S., Huen, D., Rowe, M. et al. (1993) Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proceedings of the National Academy of Science USA 90), 8479–8483.CrossRefGoogle Scholar
  11. 11.
    Komiyama, T., Ray, C.A., Pickup, D.J. et al (1994) Inhibition of interleukin-lβ converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition. Journal of Biological Chemistry 269), 19331–19337.PubMedGoogle Scholar
  12. 12.
    Alnemri E.S., Livingston, D.J., Nicholson, D.W. et al. (1996) Human ICE/CED-3 protease nomenclature. Cell 87), 171.PubMedCrossRefGoogle Scholar
  13. 13.
    Nagata, S. and Golstein, P. (1995) The Fas death factor. Science 267), 1449–1456.PubMedCrossRefGoogle Scholar
  14. 14.
    Steller, H. (1995) Mechanisms and genes of cellular suicide. Science 267), 1445–1449.PubMedCrossRefGoogle Scholar
  15. 15.
    Thompson, C.B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267), 1456–1462.PubMedCrossRefGoogle Scholar
  16. 16.
    Fesus, L., Thomazy, V., Autuori, F. et al. (1989) Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action. Federation of Experimental Biology and Science 245), 150–154.Google Scholar
  17. 17.
    Arends, M.J., Morris, R.J. and Wyllie, A.H. (1990) Apoptosis: the role of the endonu-clease. American Journal of Pathology 136), 593–608.PubMedGoogle Scholar
  18. 18.
    Boyce, B.F., Windle, J.J., Reddy, S.V. et al. (1993) Targeting SV-40 T antigen to the osteoclast in transgenic mice causes osteopetrosis, transformation and apoptosis of osteoclasts. Journal of Bone and Mineral Research 8, SI 18.Google Scholar
  19. 19.
    Fuller, K., Owens, J.M., Jagger, C.J. and Chambers, T.J. (1993) M-CSF suppresses osteo-clastic apoptosis and switches function from bone resorption to migration/chemotaxis. Journal of Bone and Mineral Research 8), S384.Google Scholar
  20. 20.
    Fuller, K., Owens, J.M., Jagger, C.J. et al. (1993) Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts. Journal of Experimental Medicine 178), 1733–1744.PubMedCrossRefGoogle Scholar
  21. 21.
    Williams, G.T., Smith, CA., Spooncer, E. et al. (1990) Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 343), 76–79.PubMedCrossRefGoogle Scholar
  22. 22.
    Boyce, B.F., Wright, K., Reddy, S.V. et al. (1995) Targeting simian virus 40 T antigen to the osteoclast in transgenic mice causes osteoclast tumors and transformation and apoptosis of osteoclasts. Endocrinology 136), 5751–5759.PubMedCrossRefGoogle Scholar
  23. 23.
    Lane, D.P. and Crawford, L.V. (1979) T antigen is bound to a host protein in SV40-transformed cells. Nature 278), 261–263.PubMedCrossRefGoogle Scholar
  24. 24.
    Lowe, S.W., Schmitt, E.M., Smith, S.W. et al. (1993) p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362), 847–849.PubMedCrossRefGoogle Scholar
  25. 25.
    Liu, C.-C, Rader, J.I., Gruber, H. and Baylink, D.J. (1982) Acute reduction in osteoclast number during bone repletion. Metabolic Bone Disease and Related Research 4), 201–209.CrossRefGoogle Scholar
  26. 26.
    Liu, C.-C. and Howard, G.A. (1991) Bone-cell changes in estrogen-induced bone-mass increase in mice: dissociation of osteoclasts from bone surfaces. Anatomical Record 229), 240–250.PubMedCrossRefGoogle Scholar
  27. 27.
    Rowe, D.J. and Hausmann, R. (1976) The alteration in osteoclast morphology by diphosphonates in bone organ culture. Calcified Tissue Research 20), 53–60.PubMedCrossRefGoogle Scholar
  28. 28.
    Flanagan, A.M. and Chambers, T.J. (1989) Dichloromethylene bisphosphonate (C12MBP) inhibits bone resorption through injury to osteoclasts that resorb C12MBP-coated bone. Bone and Mineral 6), 33–43.PubMedCrossRefGoogle Scholar
  29. 29.
    Sato, M. and Grasser, W. (1990) Effects of bisphosphonates on isolated rat osteoclasts as examined by reflected light microscopy. Journal of Bone and Mineral Research 5), 31–40.PubMedGoogle Scholar
  30. 30.
    Boyce, B.F., Aufdemorte, T.B., Garrett, I.R. et al. (1989) Effects of interleukin-1 on bone turnover in normal mice. Endocrinology 125), 1142–1150.PubMedGoogle Scholar
  31. 31.
    Hughes, D.E., Wright, K.R., Mundy, G.R. and Boyce, B.F. (1995) Association of osteoclast apoptosis with loss of adhesion. Journal of Bone and Mineral Research 10), S326.Google Scholar
  32. 32.
    Seiander, K.S., Monkkonen, J., Karhukorpi, E-K. et al (1996) Characteristics of clodronate-induced apoptosis in osteoclasts and macrophages. Molecular Pharmacology 50), 1127–1138.Google Scholar
  33. 33.
    Wright, K.R., Hughes, D.E., Guise, T.A. et al. (1994) Osteoclasts undergo apoptosis at the interface between resorption and formation in bone remodelling units. Journal of Bone and Mineral Research 9), SI 74.Google Scholar
  34. 34.
    Parfitt, A.M., Mundy, G.R., Roodman, G.D. et al. (1996) A new model for the regulation of bone resorption, with particular reference to the effects of bisphosphonates. Journal of Bone and Mineral Research 11), 150–159.PubMedGoogle Scholar
  35. 35.
    Hughes, D.E., Wright, K.R., Uy, H.L. et al. (1995) Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. Journal of Bone and Mineral Research 10), 1478–1487.PubMedGoogle Scholar
  36. 36.
    Kameda, T., Ishikawa, H. and Tsutsui, T. (1995) Detection and characterization of apoptosis in osteoclasts in vitro. Biochemical and Biophysical Research Communications 207), 753–760.PubMedCrossRefGoogle Scholar
  37. 37.
    Kameda, T., Miyazawa, K., Yoshihisa, M. et al (1996) Vitamin K2 inhibits osteoclastic bone resorption by inducing osteoclast apoptosis. Biochemical and Biophysical Research Communications 220), 515–519.PubMedCrossRefGoogle Scholar
  38. 38.
    Lutton, J.D., Moonga, B.S. and Dempster, D.W. (1996) Osteoclast demise in the rat: physiological versus degenerative cell death. Experimental Physiology 81), 251–260.PubMedGoogle Scholar
  39. 39.
    Arnett, T.R., Lindsay, R., Kilb, J.M. et al (1996) Selective toxic effects of tomoxifen on osteoclasts: comparison with the effects of estrogen. Journal of Endocrinology 149, 503-508.Google Scholar
  40. 40.
    Hughes, D.E., Dai, A., Tiffee, J.C. et al (1996) Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-β. Nature Medicine 2), 1132–1136.PubMedCrossRefGoogle Scholar
  41. 41.
    Hughes, D.E., Jilka, R., Manolagas, S. et al (1995) Sex steroids promote osteoclast apoptosis in vitro and in vivo. Journal of Bone and Mineral Research 10), S150.Google Scholar
  42. 42.
    Horowitz, M.C. (1993) Cytokines and estrogen in bone: anti-osteoporotic effects. Science 260), 626–627.PubMedCrossRefGoogle Scholar
  43. 43.
    Pacifici, R. (1996) Estrogen, cytokines, and pathogenesis of postmenopausal ostteo-porosis. Journal of Bone and Mineral Research 11, 1043–1051.Google Scholar
  44. 44.
    Hughes, D.E., Wright, K.R., Mundy, G.R. and Boyce, B.F. (1994) TGFβ1 induces osteoclast apoptosis in vitro. Journal of Bone and Mineral Research 9), S138.Google Scholar
  45. 45.
    Takahashi, N., Yamana, H., Yoshiki, S. et al (1988) Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 122), 1373–1382.PubMedCrossRefGoogle Scholar
  46. 46.
    Oursler, M.J., Pederson, L., Fitzpatrick, L. and Speisberg, T. (1996) Human giant cell tumors of bone (osteoclastomas) are estrogen target cells. Proceedings of the National Academy of Science USA 91), 5227–5231.CrossRefGoogle Scholar
  47. 47.
    Parfitt, A.M. (1994) Osteonal and hemi-osteonal remodeling: the spatial and temporal framework for signal traffic in adult human bone. Journal of Cell Biochemistry 55), 273–286.CrossRefGoogle Scholar
  48. 48.
    Parfitt, A.M., Villanueva, A.R., Foldes, J. and Rai, D.S. (1995) Relations between histologic indices of bone formation: implications for the pathogenesis of spinal osteoporosis. Journal of Bone and Mineral Research 10), 466–473.PubMedGoogle Scholar
  49. 49.
    McCabe, L.R., Kockx, M., Lian, J. et al (1995) Selective expression of fos-and jun-related genes during osteoblast proliferation and differentiation. Experimental Cell Research 218), 255–262.PubMedCrossRefGoogle Scholar
  50. 50.
    Kitajima, I., Nakajima, T., Imamura, T. et al (1996) Induction of apoptosis in murine clonal osteoblasts expressed by human T-cell leukemia virus type I tax by NF-κB and TNF-α. Journal of Bone and Mineral Research 11), 200–210.PubMedGoogle Scholar
  51. 51.
    Kitajima, L, Soejima, Y., Takasaki, I. et al (1996) Ceramide-induced nuclear translocation of NF-κB is a potential mediator of the apoptotic response to TNF-α in murine clonal osteoblasts. Bone 19), 263–269.PubMedCrossRefGoogle Scholar
  52. 52.
    Bursch, W., Lauer, B., Timmermann-Trosiener, I. et al. (1984) Controlled death (apop-tosis) of normal and putative preneoplastic cells in rat liver following withdrawal of tumor promoters. Carcinogenesis 5, 453–458.PubMedCrossRefGoogle Scholar
  53. 53.
    Lee, F.D. (1993) Importance of apoptosis in the histopathology of drug related lesions in the large intestine. Journal of Clinical Pathology 46), 118–122.PubMedGoogle Scholar
  54. 54.
    Jande, S.S. and Bélanger, L.F. (1971) Electron microscopy of osteocytes and the peri-cellular matrix in rat trabecular bone. Calcified Tissue Research 6), 280–289.PubMedCrossRefGoogle Scholar
  55. 55.
    Tonna, E.A. (1972) An electron microscopical study of osteocyte release during osteoclasis in mice at different ages. Clinical Orthopedics and Related Research 87), 311–317.CrossRefGoogle Scholar
  56. 56.
    Aarden, E.M., Burger, E.H. and Nijweide, P.J. (1994) Functions of osteocytes in bone. Journal of Cellular Biochemistry 55), 287–299.PubMedCrossRefGoogle Scholar
  57. 57.
    Elmardi, A.S., Katchburian, M.V. and Katchburian, E. (1990) Electron microscopy of developing calvaria reveals images that suggest that osteoclasts engulf and destroy osteocytes during bone resorption. Calcified Tissue International 46), 239–245.PubMedCrossRefGoogle Scholar
  58. 58.
    Bronckers, A.L.J.J., Goei, W., Luo, G. et al. (1996) DNA fragmentation during bone formation in neonatal rodents assessed by transferase-mediated end labeling. Journal of Bone and Mineral Research 11), 1281–1291.PubMedCrossRefGoogle Scholar
  59. 59.
    Dunstan, C.R., Evans, R.A., Hills, E. et al (1990) Bone death in hip fracture in the elderly. Calcified Tissue International 47), 270–275.PubMedCrossRefGoogle Scholar
  60. 60.
    Noble, B.S., Stevens, H., Loveridge, N. and Reeve, J. (1997) Identification of apoptotic changes in osteocytes in normal and pathological human bone. Bone 20), 273–282.PubMedCrossRefGoogle Scholar
  61. 61.
    Lewinson, D. and Silbermann, M. (1992) Chondroclasts and endothelial cells collaborate in the process of cartilage resorption. Anatomical Record 233), 504–514.PubMedCrossRefGoogle Scholar
  62. 62.
    Roach, H.I., Erenpreisa, J. and Aigner, T. (1995) Osteogenic differentiation of hyper-trophic chondrocytes involves cell divisions and apoptosis. Journal of Cell Biology 131), 483–494.PubMedCrossRefGoogle Scholar
  63. 63.
    Vortkamp A., Lee, K., Lanske, B. et al. (1996) Regulation of rate of cartilage differentiation by Indian hedgehog and FTH-related protein. Science 273), 613–621.PubMedCrossRefGoogle Scholar
  64. 64.
    Lanske, B., Karaplis, A.C., Lee, K. et al (1996) PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science 273), 663–666.PubMedCrossRefGoogle Scholar
  65. 65.
    Amling, M., Neff, L., Tanaka, S. et al (1997) Bcl-2 lies downstream of parathyroid hormone-related peptide in a signalling pathway that regulates chondrocyte maturation during skeletal development. Journal of Cell Biology 136), 205–213.PubMedCrossRefGoogle Scholar
  66. 66.
    Wyllie, A.H. (1994) Death from inside out: an overview. Philosophical Transactions of the Royal Society of London 345, 237–241.CrossRefGoogle Scholar
  67. 67.
    Olive, P.L, Wlodek, D. and Banath, J.P. (1991) DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. Cancer Research 51), 4671–4676.PubMedGoogle Scholar
  68. 68.
    Ostling, O. and Johanson, J.P. (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochemical and Biophysical Research Communications 123), 291–298.PubMedCrossRefGoogle Scholar
  69. 69.
    Galotto, M., Campanile, G., Robino, G. et al (1994) Hypertrophic chondrocytes undergo further differentation to osteoblast-like cells and participate in the initial bone formation in developing chick embryo. Journal of Bone and Mineral Research 9), 1239–1248.PubMedGoogle Scholar
  70. 70.
    Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. Journal of Cell Biology 119), 493–501.PubMedCrossRefGoogle Scholar
  71. 71.
    Gold, R., Schmied, M., Rothe, G. et al. (1993) Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections. Journal of Histochemistry and Cytochemistry 41), 1023–1030.PubMedGoogle Scholar
  72. 72.
    Ansari, B., Coates, P.J., Greenstein, B.D. and Hall P.A. (1993) In situ end-labelling detects DNA strand breaks in apoptosis and other physiological and pathological states. Journal of Pathology 170), 1–8.PubMedCrossRefGoogle Scholar
  73. 73.
    Wright, K.R., Story, B., Hughes, D.E. et al. (1995a) Standard morphology is more sensitive than TUNEL for identification of apoptosis in osteoclasts. Journal of Bone and Mineral Research 10), S324.Google Scholar
  74. 74.
    Arndt-Jovin, D.J. and Jovin, T.M. (1977) Analysis and sorting of living cells according to deoxyribonucleic acid content. Journal of Histochemistry and Cytochemistry 25), 585–589.PubMedGoogle Scholar
  75. 75.
    Fadok, V.A., Voelker, D.R., Campbell, P.A.S. et al. (1992) Exposure of phos-phatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. Journal of Immunology 148), 2207–2216.Google Scholar
  76. 76.
    Martin, S.J., Reutelingsperger, C.P.M., McGahon, AJ. et al (1995) Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus. Inhibition of overexpression of Bcl-2 and Abl. Journal of Experimental Medicine 182), 1545–1557.PubMedCrossRefGoogle Scholar
  77. 77.
    Vermes, I., Haanen, C. and Reutelingsperger, C.P.M. (1995) A novel assay for apoptosis: Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. Journal of Immunologic Methods 184), 39–51.CrossRefGoogle Scholar

Copyright information

© Chapman and Hall Ltd 1998

Authors and Affiliations

  • Brendan F. Boyce
  • David E. Hughes
  • Kenneth R. Wright

There are no affiliations available

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