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Use of the Fibroblast Model in the Study of Cellular Senescence

  • Protocol
Aging Methods and Protocols

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 38))

Abstract

In this chapter, we present standard procedures for the culture of human cells that exhibit a finite proliferative capacity (replicative life-span). The use of a cell culture model has the advantage of providing a controlled environment to study a wide variety of cellular phenomena. It also has the inherent limitation of isolating cells from the regulatory elements that might be provided by other types of cells in vivo. Nevertheless, cell culture models have been crucial to our current understanding of mechanisms of growth, differentiation, development, and neoplasia and numerous other disease states. In this chapter we present procedures for human fibroblast culture including serumfree cultivation of cells, which is necessary when the cellular environment must be fully defined. In addition, we present procedures for the determination of replicative life-span, saturation density, and assessment of replicative capacity from labeled thymidine incorporation in fibroblasts. The methods described here have been well tested and provide highly reproducible results (1, 2).

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References

  1. Cristofalo, V. J. and Charpentier, R. (1980) A standard procedure for cultivating human diploid fibroblastlike cells to study cellular aging. J. Tissue Cult. Methods 6, 117–121.

    Article  CAS  Google Scholar 

  2. Phillips, P. D. and Cristofalo, V. J. (1989) Cell culture of human diploid fibroblasts in serum-containing and serum-free media, in Cell Growth and Division: A Practical Approach (Baserga, R., ed.), IRL, Washington, DC, 121–132.

    Google Scholar 

  3. Swim, H. E. and Parker, R. F. (1957) Culture characteristics of human fibroblasts propagated serially. Am. J. Hyg. 66, 235–243.

    PubMed  CAS  Google Scholar 

  4. Hayflick, L. and Moorhead, P. S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585–621.

    Article  Google Scholar 

  5. Cristofalo, V. J. and Pignolo, R. J. (1993) Replicative senesence of human fibroblast-like cells in culture. Physiol. Rev. 73, 617–638.

    PubMed  CAS  Google Scholar 

  6. Ponten, J. (1973) Aging properties of glia, in INSERM (Bourliere, F., Courtois, Y., Macieira-Coelho, A., and Robert, L., eds.), INSERM, Paris, pp. 53–64.

    Google Scholar 

  7. Rheinwald, J. G. and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–343.

    Article  PubMed  CAS  Google Scholar 

  8. Bierman, E. L. (1978) The effects of donor age on the in vitro life span of cultured human arterial smooth-muscle cells. In Vitro 14, 951–955.

    Article  PubMed  CAS  Google Scholar 

  9. Tassin, J., Malaise, E., and Courtois, Y. (1979) Human lens cells have an in vitro proliferative capacity inversely proportional to the donor age. Exp. Cell Res. 123, 388–392.

    Article  CAS  Google Scholar 

  10. Mueller, S. N., Rosen, E. M., and Levine, E. M. (1980) Cellular senescence in a cloned strain of bovine fetal aortic endothelial cells. Science 207, 889–891.

    Article  PubMed  CAS  Google Scholar 

  11. Tice, R. R., Schneider, E. L., Kram, D., and Thorne, P. (1979) Cytokinetic analysis of impaired proliferative response of peripheral lymphocytes from aged humans to phytohemagglutinin. J. Exp. Med. 149, 1029–1041.

    Article  PubMed  CAS  Google Scholar 

  12. Le Guill, Y., Simon, M., Lenoir, P., and Bourel, M. (1973) Long-term culture of human adult liver cells: morphological changes related to in vitro senescence and effect of donor’s age on growth potential. Gerontologia 19, 303–313.

    Article  Google Scholar 

  13. Medrano, E. E., Yang, F., Boissy, R., Farooqui, J., Shah, V., Matsumoto, K., Nordlund, J. J., and Park, H. Y. (1994) Terminal differentiation and senescence in the human melanocyte: repression of tyrosine-phosphorylation of the extracellular signal-regulated kinase 2 selectively defines the two phenotypes. Mol. Biol. Cell 5, 497–509.

    PubMed  CAS  Google Scholar 

  14. Cristofalo, V. J., Palazzo, R., and Charpentier, R. (1980) Limited lifespan of human diploid fibroblasts in vitro: metabolic time or replications?, in Neural Regulatory Mechanisms During Aging (Adelman, R. C., Roberts, J., Baker, G. T., Baskin, S. I. and Cristofalo, V. J., eds.), Alan R. Liss, New York, pp. 203–206.

    Google Scholar 

  15. Cristofalo, V. J., Phillips, P. D., Sorger, T., and Gerhard, G. (1989) Alterations in the responsiveness of senescent cells to growth factors. J. Gerontol. 44, 55–62.

    PubMed  CAS  Google Scholar 

  16. Allsopp, R. C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E. V., Futcher, A. B., Greider, C. W., and Harley, C. B. (1992) Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89, 10,114–10,118.

    Article  PubMed  CAS  Google Scholar 

  17. Wright, W. E. and Shay, J. W. (1992) Telomere positional effects and the regulation of cellular senescence. TIG 8, 193–197.

    Article  PubMed  CAS  Google Scholar 

  18. Seshadri, T. and Campisi, J. (1990) Repression of c-fos transcription and an alteredgentic program in senescent human fibroblasts. Science 247, 205–209.

    Article  PubMed  CAS  Google Scholar 

  19. Oshima, J., Campisi, J., Tannock, C. A., and Martin, G. M. (1995) Regulation of c-fos expression in senescing Werner syndrome fibroblasts differs from that observed in senescing fibroblasts from normal donors. J. Cell. Physiol. 162, 277–283.

    Article  PubMed  CAS  Google Scholar 

  20. Keogh, B. P., Tresini, M., Cristofalo, V. J., and Allen, R. G. (1995) Effects of cellular aging on the induction of c-fos by antioxidant treatments. Mech. Ageing Dev. 86, 151–160.

    Article  Google Scholar 

  21. Levy, M. Z., Allsopp, R. C., Futcher, A. B., Greider, C. W., and Harley, C. B. (1992) Telomere end-replication problem and cell aging. J. Mol. Biol. 225, 951–960.

    Article  PubMed  CAS  Google Scholar 

  22. von Zglinick, T., Saretzki, G., Döcke, W., and Lotze, C. (1995) Mild hyperoxia shortens telomeres and inhibits proliferation of fibroblasts: a model for senescence? Exp. Cell Res. 220, 186–192.

    Article  Google Scholar 

  23. Feng, J., Funk, W. D., Wang, S.-S., Weinrich, S. L., Avilion, A. A., Chiu, C.-C., Adams, R. R., Chang, E., Allsopp, R. C., Yu, J., Le, S., West, M. D., Harley, C. B., Andrews, W. H., Greider, C. W., and Villeponteau, B. (1995) The RNA component of human telomerase. Science 269, 1236–1241.

    Article  PubMed  CAS  Google Scholar 

  24. Counter, C. M., Avilion, A. A., LeFeuvre, C. E., Stewart, N. G., Greider, C. W., Harley, C. B., and Bacchetti, S. (1992) Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11, 1921–1929.

    PubMed  CAS  Google Scholar 

  25. Blasco, M. A., Funk, W., Villeponteau, B., and Greider, C. W. (1995) Functional characterization and developmental regulation of mouse telomerase RNA. Science 269, 1267–1270.

    Article  PubMed  CAS  Google Scholar 

  26. Wainwright, L. J., Middleton, P. G., and Rees, J. L. (1995) Changes in mean telomere length in basal cell carcinomas of the skin. Genes Chromosomes Cancer 12, 45–49.

    Article  PubMed  CAS  Google Scholar 

  27. Hiyama, K., Ishioka, S., Shirotani, Y., Inai, K., Hiyama, E., Murakami, I., Isobe, T., Inamizu, T., and Yamakido, M. (1995) Alterations in telomeric repeat length in lung cancer are associated with loss of heterozygosity in p53 and Rb. Oncogene 10, 937–944.

    PubMed  CAS  Google Scholar 

  28. Goldstein, S. and Harley, C. B. (1979) In vitro studies of age-associated diseases. Fed. Proc. 38, 1862–1867.

    PubMed  CAS  Google Scholar 

  29. Brown, W. T. (1990) Genetic diseases of premature aging as models of senescence, in Annual Review of Gerontology and Geriatrics, vol. 10 (Cristofalo, V. J. and Lawton, M. P., eds.), Springer, New York, pp. 23–52.

    Google Scholar 

  30. Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C.-P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S., and Wright, W. E. (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352.

    Article  PubMed  CAS  Google Scholar 

  31. Harley, C. B., Shmookler Rei, R. J., and Goldstein, S. (1982) Loss of repetitious DNA in proliferating somatic cells may be due to unequal recombination. J. Theor. Biol. 94, 1–12.

    Article  PubMed  CAS  Google Scholar 

  32. Shmookler Rei, R. J. and Goldstein, S. (1980) Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts. Cell 21, 739–749.

    Article  Google Scholar 

  33. Gorman, S. D. and Cristofalo, V. J. (1985) Reinitiation of cellular DNA synthesis in BrdU-selected nondividing senescent WI-38 cells by simian virus 40 infection. J. Cell. Physiol. 125, 122–126.

    Article  PubMed  CAS  Google Scholar 

  34. Chang, C.-D., Phillips, P., Lipson, K. E., Cristofalo, V. J., and Baserga, R. (1991) Senescent human fibroblasts have a post-transcriptional block in the expression of the proliferating cell nuclear antigen gene. J. Biol. Chem. 266, 8663–8666.

    PubMed  CAS  Google Scholar 

  35. Pendergrass, W. R., Saulewicz, A. C., Hanaoka, F., and Norwood, T. H. (1994) Murine temperature-sensitive DNA polymerase alpha mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in heterokaryons at the nonpermissive condition. J. Cell. Physiol. 158, 270–276.

    Article  PubMed  CAS  Google Scholar 

  36. Pendergrass, W. R., Angello, J. C., Saulewicz, A. C., and Norwood, T. H. (1991) DNA polymerase alpha and the regulation of entry into S phase in heterokaryons. Exp. Cell Res. 192, 426–432.

    Article  PubMed  CAS  Google Scholar 

  37. Pendergrass, W. R., Angello, J. C., Kirschner, M. D., and Norwood, T. H. (1991) The relationship between the rate of entry into S phase, concentration of DNA polymerase alpha, and cell volume in human diploid fibroblast-like monokaryon cells. Exp. Cell Res. 192, 418–425.

    Article  PubMed  CAS  Google Scholar 

  38. Cristofalo, V. J. (1973) Cellular senescence, factors modulating cell proliferation in vitro, in INSERM (Bourliere, F., Courtois, Y., Maciera-Coelho, A., and Robert, L., eds.), INSERM, Paris, pp. 65–92.

    Google Scholar 

  39. Olashaw, N. E., Kress, E. D., and Cristofalo, V. J. (1983) Thymidine triphosphate synthesis in senescent WI38 cells. Relationship to loss of replicative capacity. Exp. Cell Res. 149, 547–554.

    Article  PubMed  CAS  Google Scholar 

  40. Pignolo, R. J., Martin, B. G., Horton, J. H., Kalbach, A. N., and Cristofalo, V. J. (1998) The pathway of cell senescence: WI-38 cells arrest in late G1 and are unable to traverse the cell cycle from a true G0 state. Exp. Gerontol. 33, 67–80.

    Article  PubMed  CAS  Google Scholar 

  41. Rittling, S. R., Brooks, K. M., Cristofalo, V. J., and Baserga, R. (1986) Expression of cell cycle-dependent genes in young and senescent WI-38 fibroblasts. Proc. Natl. Acad. Sci. USA 83, 3316–3320.

    Article  PubMed  CAS  Google Scholar 

  42. Stein, G. H., Drullinger, L. F., Robetorye, R. S., Pereira-Smith, O. M., and Smith, J. R. (1991) Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation. Proc. Natl. Acad. Sci. USA 88, 11,012–11,016.

    Article  PubMed  CAS  Google Scholar 

  43. Cristofalo, V. J., Doggett, D. L., Brooks-Frederich, K. M., and Phillips, P. D. (1989) Growth factors as probes of cell aging. Exp. Gerontol. 24, 367–374.

    Article  PubMed  CAS  Google Scholar 

  44. Hosokawa, M., Phillips, P. D., and Cristofalo, V. J. (1986) The effect of dexamethasone on epidermal growth factor binding and stimulation of proliferation in young and senescent WI38 cells. Exp. Cell Res. 164, 408–414.

    Article  PubMed  CAS  Google Scholar 

  45. De Tata, V., Ptasznik, A., and Cristofalo, V. J. (1993) Effect of the tumor promotor phorbol 12-myristate 13-acetate (PMA) on proliferation of young and senescent WI-38 human diploid fibroblasts. Exp. Cell Res. 205, 261–269.

    Article  PubMed  Google Scholar 

  46. Goodman, L. and Stein, G. H. (1994) Basal and induced amounts of interleukin 6 mRNA decline progressively with age in human fibroblasts. J. Biol. Chem. 269, 19250–19255.

    PubMed  CAS  Google Scholar 

  47. Wheaton, K., Atadja, P., and Riabowol, K. (1996) Regulation of transcription factor activity during cellular aging. Biochem. Cell Biol. 74, 523–534.

    Article  PubMed  CAS  Google Scholar 

  48. Hara, E., Uzman, J. A., Dimri, G. P., Nehlin, J. O., Testori, A., and Campisi, J. (1996) The helix-loop-helix protein Id-1 and a retinoblastoma protein binding mutant of SV40 T antigen synergize to reactivate DNA synthesis in senescent human fibroblasts. Dev. Genet. 18, 161–172.

    Article  PubMed  CAS  Google Scholar 

  49. Gerhard, G. S., Phillips, P. D., and Cristofalo, V. J. (1991) EGF-and PDGF-stimulated phosphorylation in young and senescent WI-38 cells. Exp. Cell Res. 193, 87–92.

    Article  PubMed  CAS  Google Scholar 

  50. Phillips, P. D., Kuhnle, E., and Cristofalo, V. J. (1983) 125I-EGF binding activity is stable throughout the replicative life-span of WI-38 cells. J. Cell. Physiol. 114, 311–316.

    Article  PubMed  CAS  Google Scholar 

  51. Phillips, P. D., Pignolo, R. J., and Cristofalo, V. J. (1987) Insulin-like growth factor-I: specific binding to high and low affinity sites and mitogenic action throughout the life span of WI-38 cells. J. Cell. Physiol. 133, 135–143.

    Article  PubMed  CAS  Google Scholar 

  52. Ferber, A., Chang, C., Sell, C., Ptasznik, A., Cristofalo, V. J., Hubbard, K., Ozer, H. L., Adamo, M., Roberts, C. T., Jr., LeRoith, D. (1993) Failure of senescent human fibroblasts to express the insulin-like growth factor-1 gene. J. Biol. Chem. 268, 17,883–17,888.

    PubMed  CAS  Google Scholar 

  53. Brooks-Frederich, K. M., Cianciarulo, F. L., Rittling, S. R., and Cristofalo, V. J. (1993) Cell cycle-dependent regulation of Ca2+ in young and senescent WI-38 cells. Exp. Cell Res. 205, 412–415.

    Article  PubMed  CAS  Google Scholar 

  54. Allen, R. G., Tresini, M., Keogh, B. P., Doggett, D. L., and Cristofalo, V. J. (1999) Differences in electron transport potential antioxidant defenses and oxidant generation in young and senescent fetal lung fibroblasts (WI-38). J. Cell. Physiol. 180, 114–122.

    Article  PubMed  CAS  Google Scholar 

  55. Blumenthal, E. J., Miller, A. C., Stein, G. H., and Malkinson, A. M. (1993) Serine/ threonine protein kinases and calcium-dependent protease in senescent IMR-90 fibroblasts. Mech. Ageing Dev. 72, 13–24.

    Article  PubMed  CAS  Google Scholar 

  56. Venable, M. E., Blobe, G. C., and Obeid, L. M. (1994) Identification of a defect in the phospholipase D/diacylglycerol pathway in cellular senescence. J. Biol. Chem. 269, 26,040–26,044.

    PubMed  CAS  Google Scholar 

  57. Cook, S. J. and McCormick, F. (1996) Kinetic and biochemical correlation between sustained p44ERK1 (44 kDa extracellular signal-regulated kinase 1) activation and lysophosphatidic acid-stimulated DNA synthesis in Rat-1 cells. Biochem. J. 320, 237–245.

    PubMed  CAS  Google Scholar 

  58. Lavoie, J. N., G, L. A., Brunet, A., Muller, R., and Pouyssegur, J. (1996) Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway. J. Biol. Chem. 271, 20608–20616.

    Article  PubMed  CAS  Google Scholar 

  59. Meloche, S. (1995) Cell cycle reentry of mammalian fibroblasts is accompanied by the sustained activation of p44mapk and p42mapk isoforms in the G1 phase and their inactivation at the G1/S transition. J. Cell. Physiol. 163, 577–588.

    Article  PubMed  CAS  Google Scholar 

  60. Walter, S. A., Guadagno, T. M., and Ferrell, J. E., Jr. (1997) Induction of a G2-phase arrest in Xenopus egg extracts by activation of p42 mitogen-activated protein kinase. Mol. Biol. Cell 8, 2157–2169.

    PubMed  CAS  Google Scholar 

  61. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., and Lowe, S. W. (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16ink4a. Cell 88, 593–602.

    Article  PubMed  CAS  Google Scholar 

  62. Rohme, D. (1981) Evidence for a relationship between longevity of mammalian species and life spans of normal fibroblasts in vitro and erythrocytes in vivo. Proc. Natl. Acad. Sci. USA 78, 5009–5013.

    Article  PubMed  CAS  Google Scholar 

  63. Hayflick, L. (1965) The limited in vitro lifetime of human diploid strains. Exp. Cell Res. 37, 614–636.

    Article  PubMed  CAS  Google Scholar 

  64. Schneider, E. L. and Mitsui, Y. (1976) The relationship between in vitro cellular aging and in vivo human age. Proc. Natl. Acad. Sci. USA 73, 3584–3588.

    Article  PubMed  CAS  Google Scholar 

  65. Martin, G. M., Sprague, C. A., and Epstein, C. J. (1970) Replicative life-span of cultivated human cells. Lab. Invest. 23, 86–92.

    PubMed  CAS  Google Scholar 

  66. Guilly, Y. L., Simon, M., Lenoir, P., and Bourel, M. (1973) Long-term culture of human adult liver cells: morphological changes related to in vitro senescence and effect of donor’s age on growth potential. Gerontoglia (Basel) 19, 303–313.

    Article  Google Scholar 

  67. Bruce, S. A., Deamond, S. F., and Ts’o, P. O. P. (1986) In vitro senescence of syrian hamster mesenchymal cells of fetal to aged adult origin, inverse relationship between in vivo donor age and in vitro proliferative capacity. Mech. Ageing Dev. 34, 151–173.

    Article  PubMed  CAS  Google Scholar 

  68. Goldstein, S., Moerman, E. J., Soeldner, J. S., Gleason, R. E., and Barnett, D. M. 1978) Chronologic and physiologic age affect replicative life-span of fibroblasts rom diabetic, prediabetic, and normal donors. Science 199, 781–782.

    Google Scholar 

  69. Schneider, E. L. (1979) Cell replication and aging: in vitro and in vivo studies. Fed. roc. 38, 1857–1861.

    CAS  Google Scholar 

  70. Smith, J. R., Pereira-Smith, O. M., and Schneider, E. L. (1978) Colony size distriution as a measure of in vivo and in vitro aging. Proc. Natl. Acad. Sci. USA 75, 353–1356.

    Google Scholar 

  71. Goldstein, S., Littlefield, J. W., and Soeldner, J. S. (1969) Diabetes mellitus and ging: diminished plating efficiency of cultured human fibroblasts. Proc. Natl. cad. Sci. USA 64, 155–160.

    Article  CAS  Google Scholar 

  72. Martin, G. M. (1978) Genetic syndromes in man with potential relevance to athobiology of aging, in Genetic Effects on Aging (Bergsma, D. and Harrison, D.., eds.), Alan Liss, New York, pp. 5–39.

    Google Scholar 

  73. Danes, B. S. (1971) Progeria: a cell culture study on aging. J. Clin. Invest. 50, 000–2003.

    Article  CAS  Google Scholar 

  74. Schneider, E. L. and Epstein, C. J. (1972) Replication rate and lifespan of cultured ibroblasts in Down’s syndrome. Proc. Soc. Exp. Biol. Med. 141, 1092–1094.

    PubMed  CAS  Google Scholar 

  75. Martin, G. M., Ogburn, C. E., and Sprague, C. A. (1981) Effects of age on cell division capacity, in Aging: A Challenge to Science and Society, vol. 1 (Danon, D., Shock, N. W., and Marios, M., eds.), Oxford University Press, Oxford, England, pp. 124–135.

    Google Scholar 

  76. Rubin, H. (1997) Cell aging in vivo and in vitro. Mech. Ageing Dev. 98, 1–35.

    Article  PubMed  CAS  Google Scholar 

  77. Pignolo, R. J., Masoro, E. J., Nichols, W. W., Brandt, C. I., and Cristofalo, V. J. (1992) Skin fibroblasts from aged Fischer 344 rats undergo similar changes in replicative life span but not immortalization with caloric restriction of donors. Exp. Cell Res. 201, 16–22.

    Article  PubMed  CAS  Google Scholar 

  78. Bruce, S. A. and Deamond, S. F. (1991) Longitudinal study of in vivo wound repair and in vitro cellular senescence of dermal fibroblasts. Exp. Gerontol. 26, 17–27.

    Article  PubMed  CAS  Google Scholar 

  79. Deamond, S. F. and Bruce, S. A. (1991) Age-related differences in the promoterinduced extension of in vitro proliferative life span of syrian hamster fibroblasts. Mech. Ageing Dev. 60, 143–152.

    Article  PubMed  CAS  Google Scholar 

  80. Cristofalo, V. J., Allen, R. G., Pignolo, R. P., Martin, B. M., and Beck, J. C. (1998) Relationship between donor age and the replicative life spans of human cells in culture: a re-evaluation. Proc. Natl. Acad. Sci. USA 95, 10614–10619.

    Article  PubMed  CAS  Google Scholar 

  81. Millis, A. J. T., Sottile, J., Hoyle, M., Mann, D. M., and Diemer, V. (1989) Collagenase production by early and late passage cultures of human fibroblasts. Exp. Gerontol. 24, 559–575.

    Article  PubMed  CAS  Google Scholar 

  82. Sottile, J., Mann, D. M., Diemer, V., and Millis, A. J. (1989) Regulation of collagenase and collagenase mRNA production in early-and late-passage human diploid fibroblasts. J. Cell. Physiol. 138, 281–290.

    Article  PubMed  CAS  Google Scholar 

  83. Seshadri, T. and Campisi, J. (1989) Growth-factor-inducible gene expression in senescent human fibroblasts. Exp. Gerontol. 24, 515–522.

    Article  PubMed  CAS  Google Scholar 

  84. Grassilli, E., Bellesia, E., Salomoni, P., Croce, M. A., Sikora, E., Radziszewska, E., Tesco, G., Vergelli, M., Latorraca, S., Babieri, D., Fagiolo, U., Santacaterina, S., Amaducci, L., Tiozzo, R., Sorbi, S., and Franceschi, C. (1996) C-Fos/C-Jun expression and AP-1 activation in skin fibroblasts from centenarians. Biochem. Biophys. Res. Commun. 226, 517–523.

    Article  PubMed  CAS  Google Scholar 

  85. Millis, A. J., Hoyle, M., McCue, H. M., and Martini, H. (1992) Differential expression of metalloproteinase and tissue inhibitor of metalloproteinase genes in aged human fibroblasts. Exp. Cell Res. 201, 373–379.

    Article  PubMed  CAS  Google Scholar 

  86. Pignolo, R. J., Cristofalo, V. J., and Rotenberg, M. O. (1993) Senescent WI-38 cells fail to express EPC-1, a gene induced by young cells upon entry into the G0 state. J. Biol. Chem. 268, 8949–8957.

    PubMed  CAS  Google Scholar 

  87. Tresini, M., Pignolo, R., Allen, R. G., Rottenberg, M. O., and Cristofalo, V. J. (1999) Expression of EPC-1 in human skin fibroblasts derived from donors of different ages. J. Cell Physiol. 179, 11–17.

    Article  PubMed  CAS  Google Scholar 

  88. Wes, M. D., Shay, J. W., Wright, W. E., and Linskens, M. H. K. (1996) Altered expression of plasmogen activator and plasmogen activator inhibitor during cellular senescence. Exp. Gerontol. 31, 175–193.

    Article  Google Scholar 

  89. Ranby, M., Bergsdorf, N., Nisson, T., Mellbring, G., Winblad, B., and Bucht, G. (1986) Age dependence of tissue plasminogen activator concentrations in plasma, as studied by an improved enzyme-linked immunosorbent assay. Clin. Chem. 32, 2160–2165.

    PubMed  CAS  Google Scholar 

  90. Wang, S. M., Phillips, P. D., Sierra, F., and Cristofalo, V. J. (1996) Altered expression of the twist gene in young versus senescent human diploid fibroblasts. Exp. Cell Res. 228, 138–145.

    Article  PubMed  CAS  Google Scholar 

  91. Goldstein, S., Moerman, E. J., and Baxter, R. C. (1993) Accumulation of insulin-like growth factor binding protein-3 in conditioned medium of human fibroblasts increases with chronological age of donor and senescence in vitro. J. Cell. Physiol. 156, 294–302.

    Article  PubMed  CAS  Google Scholar 

  92. Keogh, B. P., Allen, R. G., Pignolo, R., Horton, J., Tresini, M., and Cristofalo, V. J. (1996) Expression of hydrogen peroxide and glutathione metabolizing enzymes in human skin fibroblasts derived from donors of different ages. J. Cell. Physiol. 167, 512–522.

    Article  PubMed  CAS  Google Scholar 

  93. Takeda, K., Gosiewska, A., and Peterkofsky, B. (1992) Similar, but not identical, modulation of expression of extracellular matrix components during in vitro and in vivo aging of human skin fibroblasts. J. Cell. Physiol. 153, 450–459.

    Article  PubMed  CAS  Google Scholar 

  94. Deguchi, Y., Negoro, S., and Kishimoto, S. (1988) Age-related changes of heat shock protein gene transcription in human peripheral blood mononuclear cells. Biochem. Biophys. Res. Commun. 157, 580–584.

    Article  CAS  Google Scholar 

  95. Kumazaki, T., Wadhwa, R., Kaul, S. C., and Mitsui, Y. (1997) Expression of endothelin, fibronectin, and mortalin as aging and mortality markers. Exp. Gerontol. 32, 95–103.

    Article  PubMed  CAS  Google Scholar 

  96. Allen, R. G., Keogh, B. P., Tresini, M., Gerhard, G. S., Volker, C., Pignolo, R. J., Horton, J., and Cristofalo, V. J. (1997) Development and age-associated differences in electron transport potential and consequences for oxidant generation. J. Biol. Chem. 272, 24,805–24,812.

    Article  PubMed  CAS  Google Scholar 

  97. Doggett, D. L., Rotenberg, M. O., Pignolo, R. J., Phillips, P. D., and Cristofalo, V. J. (1992) Differential gene expression between young and senescent, quiescent WI-38 cells. Mech. Ageing Dev. 65, 239–255.

    Article  PubMed  CAS  Google Scholar 

  98. Praeger, F. C. and Gilchrest, B. A. (1986) Influence of increased extracellular calcium and donor age on density-dependent growth inhibition of human fibroblasts (42345) Proc. Soc. Exp. Biol. Med. 182, 315–321.

    PubMed  CAS  Google Scholar 

  99. Praeger, F. C. and Cristofalo, V. J. (1986) The growth of WI-38 in a serum-free, growth factor-free, medium with elevated calcium concentrations. In Vitro 22, 355–359.

    CAS  Google Scholar 

  100. Furth, J. J., Allen, R. G., Tresini, M., Keogh, B., and Cristofalo, V. J. (1997) Abundance of α1(I) and α1(III) procollagen and p21 mRNAs in fibroblasts cultured from fetal and postnatal donors. Mech. Ageing Dev. 97, 131–142.

    Article  PubMed  CAS  Google Scholar 

  101. Nakanish, M., Robertorye, R. S., Adami, G. R., Pereira Smith, O. M., and Smith J. R. (1995) Identification of the active region of the DNA synthesis inhibitorygene p21Sdi1/CIP1/WAF1. Embo J. 14, 555–563.

    Google Scholar 

  102. Allen, R. G., Keogh, B. P., Gerhard, G., Pignolo, R., Horton, J., and Cristofalo, V. J. (1995) Expression and regulation of SOD activity in human skin fibroblasts from donors of different ages. J. Cell. Physiol. 165, 576–587.

    Article  PubMed  CAS  Google Scholar 

  103. Kumar, S., Millis, A. J. T., and Baglioni, C. (1992) Expression of interleukin 1-inducible genes and production of interleukin 1 by aging human fibroblasts. Proc. Natl. Acad. Sci. USA 89, 4683–4687.

    Article  PubMed  CAS  Google Scholar 

  104. Lapiere, C. M. (1988) Aging of fibroblasts, collagen and the dermis, in Cutaneous Aging (Kligman, A. M. and Takase, Y., eds.), University of Tokyo Press, Tokyo, 47–60.

    Google Scholar 

  105. Choi, A. M. K., Olsen, D. R., Cook, K. G., Deamond, S. F., Uitto, J., and Bruce, S. A. (1992) Differential extracellular matrix gene expression by fibroblasts during their proliferative life span in vitro and senescence. J. Cell. Physiol. 151, 147–155.

    Article  PubMed  CAS  Google Scholar 

  106. Robbins, E., Levine, E. M., and Eagle, H. (1970) Morphologic changes accompanying senescence of cultured human diploid cells. J. Exp. Med. 131, 1211–1222.

    Article  PubMed  CAS  Google Scholar 

  107. Bell, E., Marek, L. F., Levinstone, D. S., Merril, C., Sher, S., and Eden, M. (1978) Loss of division potential in vitro: aging or differentiation? Science 202, 1158–1163.

    Article  PubMed  CAS  Google Scholar 

  108. Bell, E., Marek, L., Sher, S., Merrill, C., Levinstone, D., and Young, I. (1979) Do diploid fibroblasts in culture age? Int. Rev. Cytol. (Suppl. 10), 1–9.

    Google Scholar 

  109. Bayreuther, K., Francz, P. I., Gogol, J., Maier, M., and Meinrath, G. (1991) Differentiation of primary and secondary fibroblasts in cell culture systems. Mutat. Res. 256, 233–242.

    PubMed  CAS  Google Scholar 

  110. Kontermann, K. and Bayreuther, K. (1979) The cellular aging of rat fibroblasts in vitro is a differentiation process. Gerontology 25, 261–274.

    Article  PubMed  CAS  Google Scholar 

  111. Bayreuther, K., Rodemann, H. P., Hommel, R., Dittamann, K., Albiez, M., and Francz, P. I. (1988) Human skin fibroblasts in vitro differentiate along a terminal cell lineage. Proc. Natl. Acad. Sci. USA 85, 5112–5116.

    Article  PubMed  CAS  Google Scholar 

  112. Campisi, J. (1997) Aging and cancer: the double-edged sword of replicative senescence. J. Am. Geriatr. Soc. 45, 482–488.

    PubMed  CAS  Google Scholar 

  113. Livinstone, D., Eden, M., and Bell, E. (1983) Similarity of sister-cell trajectories in fibroblast clones. J. Cell Sci. 59, 105–119.

    Google Scholar 

  114. Linskens, M. H. K., Fseng, J., Andrews, W. H., Enlow, B. E., Saati, S. M., Tonkin, L. A., Funk, W. D., and Villeponteau, B. (1995) Cataloging altered gene expression in yong and senescent cells using enhanced differential display. Nucleic Acids Res. 23, 3244–3251.

    Article  PubMed  CAS  Google Scholar 

  115. Kumar, S., Vinci, J. M., Millis, A. J. T., and Baglioni, C. (1993) Expression of interleukin-1α and β in early passage fibroblasts from aging individuals. Exp. Gerontol. 28, 505–513.

    Article  PubMed  CAS  Google Scholar 

  116. Karlsson, C. and Paulsson, Y. (1994) Age-related induction of platelet-derived growth factor A-chain mRNA in normal human fibroblasts. J. Cell. Physiol. 158, 256–262.

    Article  PubMed  CAS  Google Scholar 

  117. Slayback, J. R., Cheung, L. W., and Geyer, R. P. (1977) Comparative effects of human platelet growth factor on the growth and morphology of human fetal and adult diploid fibroblasts. Exp. Cell Res. 110, 462–466.

    Article  PubMed  CAS  Google Scholar 

  118. Furth, J. J. (1991) The steady state levels of type I collagen mRNA are reduced in senescent fibroblasts. J. Gerontol. 46, B122–B124.

    PubMed  CAS  Google Scholar 

  119. Wharton, W. (1984) Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements. Exp. Cell Res. 154, 310–314.

    Article  PubMed  CAS  Google Scholar 

  120. Clemmons, D. R. (1983) Age-dependent production of a competent factor by human fibroblasts. J. Cell. Physiol. 114, 61–67.

    Article  PubMed  CAS  Google Scholar 

  121. Kondo, H. and Yonezawa, Y. (1995) Fetal-adult phenotype transition, in terms of the serum dependency and growth factor requirements, of human skin fibroblast migration. Exp. Cell Res. 220, 501–504.

    Article  PubMed  CAS  Google Scholar 

  122. Chen, Q., Fisher, A., Reagan, J. D., Yan, L. J., and Ames, B. N. (1995) Oxidative DNA damage and senescence of human diploid fibroblasts. Proc. Natl. Acad. Sci. USA 92, 4337–4341.

    Article  PubMed  CAS  Google Scholar 

  123. Venable, M. E., Lee, J. Y., Smyth, M. J., Bielawska, A., and Obeid, L. M. (1995) Role of ceramide in cellular senescence. J. Biol. Chem. 270, 30701–30708.

    Article  PubMed  CAS  Google Scholar 

  124. Tresini, M., Mawaldewan, M., Cristofalo, V. J., and Sell, C. (1998) A phosphatidylinositol 3-kinase inhibitor induces a senescent-like growth arrest in human diploid fibroblasts. Cancer Res. 58, 1–4.

    PubMed  CAS  Google Scholar 

  125. Ogryzko, V. V., Hirai, T. H., Russanova, V. R., Barbie, D. A., and Howard, B. H. (1996) Human fibroblast commitment to a senescence-like state in response to histone deacetylase inhibitors is cell cycle dependent. Mol. Cell. Biol. bd16, 5210–5218.

    Google Scholar 

  126. Goldstein, S., Moerman, E. J., Soeldner, J. S., Gleason, R. E., and Barnett, D. M. (1979) Diabetes mellitus and genetic prediabetes. Decreased replicative capacity of cultured skin fibroblasts. J. Clin. Invest. 63, 358–370.

    Article  PubMed  CAS  Google Scholar 

  127. Phillips, P. D. and Cristofalo, V. J. (1988) Classification system based on the functional equivalency of mitogens that regulate WI-38 cell proliferation. Exp. Cell Res. 175, 396–403.

    Article  PubMed  CAS  Google Scholar 

  128. Matsumura, T., Zerrudo, Z., and Hayflick, L. (1979) Senescent human diploid cells in culture: suvival, DNA synthesis and morphology. J. Gerontol. 34, 328–334.

    PubMed  CAS  Google Scholar 

  129. Cristofalo, V. J. and Sharf, B. B. (1973) Cellular senescence and DNA synthesis: thymidine incorporation as a measure of population age in human diploid cells. Exp. Cell Res. 76, 419–427.

    Article  PubMed  CAS  Google Scholar 

  130. Cristofalo, V. J. and Kritchevsky, D. (1965) Growth and glycolysis in the human diploid cell strain WI-38. Proc. Soc. Exp. Biol. Med. 118, 1109–1113.

    PubMed  CAS  Google Scholar 

  131. Levine, E. M. (1972) Mycoplasma contamination of animal cell cultures: a simple, rapid detection method. Exp. Cell Res. 74, 99–109.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Center for Gerontological Research, Allegheny University of the Health Sciences, Philadelphia, PA

    Vincent J. Cristofalo, Craig Volker & Robert G. Allen

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  1. Vincent J. Cristofalo
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  2. Craig Volker
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  3. Robert G. Allen
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  1. School of Biomedical Sciences, University of Ulster, Northern Ireland

    Yvonne A. Barnett  & Christopher R. Barnett  & 

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© 2000 Humana Press Inc., Totowa, NJ

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Cristofalo, V.J., Volker, C., Allen, R.G. (2000). Use of the Fibroblast Model in the Study of Cellular Senescence. In: Barnett, Y.A., Barnett, C.R. (eds) Aging Methods and Protocols. Methods in Molecular Medicine, vol 38. Humana Press. https://doi.org/10.1385/1-59259-070-5:23

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  • DOI: https://doi.org/10.1385/1-59259-070-5:23

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-582-9

  • Online ISBN: 978-1-59259-070-4

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