Comprehensive Physiology Wiley Online Library

Cell Culture as a Model

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Historical Development of Cell Cultures for the Study of Aging
2 Relevance of in Vitro Cellular Senescence to in Vivo Aging
2.1 Cell Cultures as Models
2.2 Cellular Mortality, Cellular Clocks, and Death as an End Point for Aging
2.3 Aging and Evolution
2.4 In Vivo–In Vitro Parallel Changes
3 Caveats in the Use of Cell Cultures as Models
4 Cellular and Molecular Markers of Senescence in Vitro and in Vivo
5 Mechanistic Studies of in Vitro Replicative Senescence
5.1 Stochastic Mechanisms
5.2 Signal Transduction Pathways
5.3 Genetic Mechanisms
6 Summary and Conclusions
Figure 1. Figure 1.

Heterogeneity of senescent cells in culture. Multiple parallel pathways leading to DNA synthesis, one or more of which may be blocked in senescent cells (*ART*). These block(s) may be overcome in conditionally arrested cells by a variety of manipulations at putative target sites but not in irreversibly arrested cells, except by SV40 infection. PMA, phorbol 12‐myristate 13‐acetate; HC, hydrocortisone.

Figure 2. Figure 2.

Stages in the in vitro life history of normal human diploid fibroblasts. Phase I, outgrowth from explant; phase II, period of vigorous proliferation; phase III, decline in proliferative capacity; phase IV, cessation of proliferation and cell death; phase V, emergence of a stable, viable, but essentially nonproliferating cell population. Dotted line indicates the possibility, as a rare event, phase II cells may transform into an immortalized population acquiring an indefinite proliferation potential.

Figure 3. Figure 3.

Representation of growth states available to young and senescent fibroblasts and the molecular markers that define each state. Gs, senescence‐specific growth state; τ, putative transitional pathways by which conditionally arrested cells may re‐enter cell cycle;?, pathways by which young cells become senescent by an unknown mechanism(s).



Figure 1.

Heterogeneity of senescent cells in culture. Multiple parallel pathways leading to DNA synthesis, one or more of which may be blocked in senescent cells (*ART*). These block(s) may be overcome in conditionally arrested cells by a variety of manipulations at putative target sites but not in irreversibly arrested cells, except by SV40 infection. PMA, phorbol 12‐myristate 13‐acetate; HC, hydrocortisone.



Figure 2.

Stages in the in vitro life history of normal human diploid fibroblasts. Phase I, outgrowth from explant; phase II, period of vigorous proliferation; phase III, decline in proliferative capacity; phase IV, cessation of proliferation and cell death; phase V, emergence of a stable, viable, but essentially nonproliferating cell population. Dotted line indicates the possibility, as a rare event, phase II cells may transform into an immortalized population acquiring an indefinite proliferation potential.



Figure 3.

Representation of growth states available to young and senescent fibroblasts and the molecular markers that define each state. Gs, senescence‐specific growth state; τ, putative transitional pathways by which conditionally arrested cells may re‐enter cell cycle;?, pathways by which young cells become senescent by an unknown mechanism(s).

References
 1. Absher, P. M., and R. G. Absher. Clonal variation and aging of diploid fibroblasts. Exp. Cell Res. 103: 247–255, 1976.
 2. Absher, P. M., R. G. Absher, and W. D. Barnes. Genealogies of clones of diploid fibroblasts. Cinemicrophotographic observations of cell division patterns in relation to population age. Exp. Cell Res. 88: 95–104, 1974.
 3. Afshari, C. A., P. J. Vojta, L. A. Annab, P. A. Futreal, T. B. Willard, and J. C. Barrett. Investigation of the role of G1/S cell cycle mediators in cellular senescence. Exp. Cell Res. 209: 231–237, 1993.
 4. Albright, J. W., and T. Makinodan. Decline in the growth potential of spleen‐colonizing bone marrow stem cells of long‐lived aging mice. J. Exp. Med. 144: 1204–1213, 1976.
 5. Allen, R. G., and A. K. Balin. Developmental changes in the superoxide dismutase activity of human skin fibroblasts are maintained in vitro and are not caused by oxygen. J. Clin. Invest. 82: 731–734, 1988.
 6. Angello, J. C., W. R. Pendergrass, T. H. Norwood, and J. Prothero. Proliferative potential of human fibroblasts: an inverse dependence on cell size. J. Cell. Physiol. 132: 125–130, 1987.
 7. Angello, J. C., W. R. Pendergrass, T. H. Norwood, and J. Prothero. Cell enlargement: one possible mechanism underlying cellular senescence. J. Cell. Physiol. 140: 288–294, 1989.
 8. Aronson, J. F., J. W. McClaskey, and V. J. Cristofalo. Human fetal lung fibroblasts: observations on origin and stability in culture. Mech. Ageing Dev. 21: 229–244, 1983.
 9. Atillasoy, E., and P. R. Holt. Gastrointestinal proliferation and aging. J. Gerontol. 48: B43–B49, 1993.
 10. Aune, T. M., and S. L. Pogue. Generation and characterization of continuous lines of CD8+ suppressor T lymphocytes. J. Immunol. 142: 3731–3739, 1989.
 11. Balin, A. K., D. B. P. Goodman, H. Rasmussen, and V. J. Cristofalo. The effect of oxygen tension on the growth and metabolism of WI‐38 cells. J. Cell. Physiol. 89: 235–250, 1976.
 12. Balin, A. K., D. B. P. Goodman, H. Rasmussen, and V. J. Cristofalo. The effect of oxygen and vitamin E on the life span of human diploid cells in vitro. J. Cell Biol. 74: 58–67, 1977.
 13. Bayreuther, K., P. I. Francz, J. Gogol, M. Maier, and H. G. Meinrath. Differentiation of primary and secondary fibroblasts in cell culture systems. Mutat. Res. 256: 233–242, 1991.
 14. Bemiller, P. M., and L. H. Lee. Nucleolar changes in senescing WI‐38 cells. Mech. Ageing Dev. 8: 417–427, 1976.
 15. Bemiller, P. M., and J. E. Miller. Cytological changes in senescing WI‐38 cells: a statistical analysis. Mech. Ageing Dev. 10: 1–15, 1979.
 16. Bierman, E. L. The effect of donor age on the in vitro lifespan of cultured human arterial smooth‐muscle cells. In Vitro Cell. Dev. Biol. 14: 951–955, 1978.
 17. Blumenthal, E. J., A. C. K. Miller, G. H. Stein, and A. M. Malkinson. Serine/threonine protein kinases and calcium‐dependent protease in senescent IMR‐90 fibroblasts. Mech. Ageing Dev. 72: 13–24, 1993.
 18. Bowman, P. D., and C. W. Daniel. Aging of human fibroblasts in vitro: surface features and behavior of aging WI‐38 cells. Mech. Ageing Dev. 4: 147–158, 1975.
 19. Bowman, P. D., R. L. Meek, and C. W. Daniel. Decreased synthesis of nucleolar RNA in aging human cells in vitro. Exp. Cell Res. 101: 434–437, 1976a.
 20. Bowman, P. D., R. L. Meek, and C. W. Daniel. Decreased unscheduled DNA synthesis in nondividing aged WI‐38 cells. Mech. Ageing Dev. 5: 251–257, 1976b.
 21. Bradley, M. O., L. C. Erickson, and T. W. Kohn. Normal DNA strand rejoining and absence of DNA crosslinking in progeroid and aging human cells. Mutat. Res. 37: 279–292, 1976.
 22. Brandes, D., D. G. Murphy, E. B. Anton, and S. Barnard. Ultrastructural and cytochemical changes in cultured human lung cells. J. Ultrastruct. Res. 39: 465–483, 1972.
 23. Brooks, K. M., P. D. Phillips, C. R. Carlin, B. Knowles, and V. J. Cristofalo. EGF‐dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI‐38 cells. J. Cell. Physiol. 133: 523–531, 1987.
 24. Brooks‐Frederich, K. M., F. L. Cianciarulo, S. M. Rittling, and V. J. Cristofalo. Cell cycle dependent regulation of Ca++ in young and senescent WI‐38 cells. Exp. Cell Res. 205: 412–415, 1993.
 25. Buchman, T. G., D. E. Cabin, S. Vickers, C. S. Deutschman, E. Delgado, M. M. Sussman, and G. B. Bulkley. Molecular biology of circulatory shock. Part II. Expression of four groups of hepatic genes is enhanced after resuscitation from cardiogenic shock. Surgery 108: 559–566, 1990.
 26. Buetow, D. E., Cell numbers vs. age in mammalian tissues and organs. In: CRC Handbook of Cell Biology of Aging, edited by V. J. Cristofalo. Boca Raton, FL: CRC Press, 1985, p. 1–115.
 27. Burmer, G. C., C. J. Zeigler, and T. H. Norwood. Evidence for endogenous polypeptide‐mediated inhibition of cell‐cycle transit in human diploid cells. J. Cell Biol. 94: 187–192, 1982.
 28. Carlin, C. R., P. D. Phillips, B. B. Knowles, and V. J. Cristofalo. Diminished in vitro tyrosine kinase activity of the EGF receptor of senescent human fibroblasts. Nature 306: 617–620, 1983.
 29. Carlin, C. R., P. D. Phillips, K. M. Brooks‐Frederick, S. Miller, B. B. Knowles, and V. J. Cristofalo. Cleavage of the EGF receptor by an endogenous leupeptin‐sensitive protease active in detergent extracts of senescent but not young human diploid fibroblasts. J. Cell. Physiol. 160: 427–434, 1994.
 30. Carrel, A., and M. T. Burrows. Cultivation of adult tissues and organs outside of the body. JAMA 55: 1379–1381, 1910a.
 31. Carrel, A., and M. T. Burrows. Cultivation of sarcoma outside of the body. JAMA 55: 1554, 1910b.
 32. Carrel, A., and M. T. Burrows. On the physiochemical regulation of the growth of tissues. J. Exp. Med. 13: 562–570, 1911.
 33. Carrel, A., and A. H. Ebling. Age and multiplication of fibroblasts. J. Exp. Med. 34: 599–623, 1921.
 34. Chang, C., P. D. Phillips, K. E. Lipson, V. J. Cristofalo, and R. Baserga. Senescent human fibroblasts have a post‐transcriptional block in the expression of the proliferating cell nuclear antigen. J. Biol. Chem. 266: 8663–8666, 1991.
 35. Chang, Z.‐F., and K. Y. Chen. Regulation of ornithine decarboxylase and other cell cycle‐regulated genes during senescence of IMR‐90 human diploid fibroblasts. J. Biol. Chem. 263: 11431–11435, 1988.
 36. Choudhury, G. G., V. L. Sylvia, and A. Y. Sakaguchi. Decline of signal transduction by phospholipase Cγ 1 in IMR‐90 human diploid fibroblasts at high population doubling levels. FEBS Lett. 293: 211–214, 1991.
 37. Chua, C. C., D. E. Geiman, and R. L. Ladda. Receptor for epidermal growth factor retains normal structure and function in aging cells. Mech. Ageing Dev. 34: 35–55, 1986.
 38. Cohen, S. C., and H. D. Barner. Studies on unbalanced growth in Escherichia coli. Biochemistry 40: 885–893, 1954.
 39. Comings, D. E., and T. A. Okada. Electron microscopy of human fibroblasts in tissue culture during logarithmic and confluent stages of growth. Exp. Cell Res. 61: 295–301, 1970.
 40. Cooper, J. T., and S. Goldstein. Comparative studies on human skin fibroblasts: life span and lipid metabolism in medium containing fetal bovine or human serum. In Vitro Cell. Dev. Biol. 13: 473–476, 1977.
 41. Cristofalo, V. J., Metabolic aspects of aging in diploid human cells. In: Aging in Cell and Tissue Culture, edited by E. Holeckova and V. J. Cristofalo. New York: Plenum Press, 1970, p. 83–119.
 42. Cristofalo, V. J., Animal cell cultures as a model system for the study of aging. In: Advances in Gerontological Research, edited by B. L. Strehler. New York: Academic Press, 1972, vol. 4, p. 45–79.
 43. Cristofalo, V. J., Cellular senescence: factors modulating cell proliferation in vitro. In: INSERM, edited by F. Bourliere, Y. Courtois, A. Macieira‐Coelho and L. Robert. Paris: INSERM, 1973, vol. 27, p. 65–92.
 44. Cristofalo, V. J. Cellular biomarkers of aging. Exp. Gerontol. 23: 297–305, 1988.
 45. Cristofalo, V. J. On models and the study of senescence: reflections on the state of biogerontology and a farewell. J. Gerontol. 46: B207–B208, 1991.
 46. Cristofalo, V. J., and D. Kritchevsky. Cell size and nucleic acid content in the human diploid cell line WI‐38 during aging. Med. Exp. Int. J. Exp. Med. 19: 313–320, 1969.
 47. Cristofalo, V. J., and B. B. Sharf. Cellular senescence and DNA synthesis: thymidine incorporation as a measure of population age in human diploid cells. Exp. Cell Res. 76: 419–427, 1973.
 48. Cristofalo, V. J., and B. M. Stanulis‐Praeger. Cellular senescence in vitro. In: Advances in Cell Culture, edited by K. Mararmorosch. New York: Academic Press 1982, vol. 2, p. 1–67.
 49. Cristofalo, V. J., D. L. Doggett, K. M. Brooks‐Frederich, and P. D. Phillips. Growth factors as probes of cell aging. Exp. Gerontol. 24: 367–374, 1989.
 50. Cristofalo, V. J., B. V. Howard, and D. Kritchevsky. The biochemistry of human cells in culture. In: Research Progress in Organic, Biological, and Medicinal Chemistry, edited by U. Gallo and L. Santamaria. Amsterdam: North‐Holland Publishing Company, 1970, vol. 2, p. 95–146.
 51. Cristofalo, V. J., R. Palazzo, and R. L. Charpentier. Limited lifespan of human fibroblasts in vitro: metabolic time or replications? In: Neural Regulatory Mechanisms During Aging, edited by R. C. Adelman, J. Roberts, G. T. Baker, S. I. Baskin and V. J. Cristofalo. New York: Alan R. Liss, 1980, p. 203–206.
 52. Cristofalo, V. J., N. Parris, and D. Kritchevsky. Enzyme activity during the growth and aging of human cells in vitro. J. Cell. Physiol. 69: 263–272, 1967.
 53. Cristofalo, V. J., P. D. Phillips, T. Sorger, and G. Gerhard. Alterations in the responsiveness of senescent cells to growth factors. J. Gerontol. 44: 55–62, 1989.
 54. Cristofalo, V. J., R. J. Pignolo, and M. O. Rotenberg. Molecular changes with in vitro cellular senescence. In: Aging and Cellular Defense Mechanisms, edited by C. Franceschi, G. Crepaldi, V. J. Cristofalo, and J. Vijg. New York: The New York Academy of Sciences, 1992, vol. 663, p. 187–194.
 55. Cudkowicz, G., A. C. Upton, G. M. Shearer, and W. L. Hughes. Lymphocyte contact and proliferative capacity of serially transplanted mouse bone marrow. Nature 201: 165–167, 1964.
 56. Cummings, D. J. Mitochrondrial DNA in Podospora anserina: a molecular approach to cellular senescence. Monogr. Dev. Biol. 17: 254–266, 1984.
 57. Danes, B. S. Progeria: a cell culture study on aging. J. Clin. Invest. 50: 2000–2003, 1971.
 58. Daniel, C. W., K. B. Deohme, J. T. Young, P. B. Blair, and L. J. Faulkin. The in vivo lifespan of normal and preneoplastic mouse mammary glands: a serial transplantation study. Proc. Natl. Acad. Sci. USA 61: 52–60, 1968.
 59. Deknudt, G., and A. Leonard. Aging and radiosensitivity of human somatic chromosomes. Exp. Gerontol. 12: 237–240, 1977.
 60. Dell'Orco, R. T., G. B. Mertens, and P. F. Kruse, Jr.. Doubling potential and calender time of human diploid cells in culture. Exp. Cell Res. 84: 363–366, 1974.
 61. De Tata, V., A. Ptasznik, and V. J. Cristofalo. Effects of the tumor promoting agent phorbol 12‐myristate 13‐acetate (PMA) on the proliferation of young and senescent WI‐38 human diploid fibroblasts. Exp. Cell Res. 205: 261–269, 1993.
 62. Dice, J. F. Altered intracellular protein degradation in aging: a possible cause of proliferative arrest. Exp. Gerontol. 24: 451–459, 1989.
 63. Di Paolo, B. R., R. J. Pignolo, and V. J. Cristofalo. Overexpression of the two‐chain form of cathepsin B in senescent WI‐38 cells. Exp. Cell Res. 201: 500–505, 1992.
 64. D'Mello, N. P., and S. M. Jazwinski. Telomere length constancy during aging of Saccharomyces cerevisiae. J. Bacteriol. 173: 6709–6713, 1991.
 65. Doggett, D. L., M. O. Rotenberg, R. J. Pignolo, P. D. Phillips, and V. J. Cristofalo. Differential gene expression between young and senescent quiescent WI‐38 cells. Mech. Ageing Dev. 65: 239–255, 1992.
 66. Drescher‐Lincoln, C. K., and J. R. Smith. Inhibition of DNA synthesis in senescent‐proliferating human cybrids is mediated by endogenous proteins. Exp. Cell Res. 153: 208–217, 1984.
 67. Dulic, V., W. K. Kaufmann, S. J. Wilson, T. D. Tisty, E. Lees, J. W. Harper, S. J. Elledge, and S. I. Reed. p53‐Dependent inhibition of cyclin‐dependent kinase activities in human fibroblasts during radiation‐induced G1 arrest. Cell 76: 1013–1023, 1994.
 68. Duncan, E. L., N. J. Whitaker, E. L. Moy, and R. R. Reddel. Assignment of SV40‐immortalized cells to more than one complementation group for immortalization. Exp. Cell Res. 205: 337–344, 1993.
 69. Ebeling, A. H. The permanent life of connective tissue outside of the organism. J. Exp. Med. 17: 273–285, 1913.
 70. Ebeling, A. H. Measurement of the growth of tissues in vitro. J. Exp. Med. 34: 231–243, 1921.
 71. Egilmez, N. K., J. B. Chen, and S. M. Jazwinski. Specific alterations in transcript prevalence during the yeast lifespan. J. Biol. Chem. 264: 14312–14317, 1989.
 72. Egilmez, N. K., J. B. Chen, and S. M. Jazwinski. Preparation and partial characterization of old yeast cells. J. Gerontol. 45: B9–B17, 1990.
 73. Fairweather, S., M. Fox, and B. P. Margison. The in vitro lifespan of MRC‐5 cells is shortened by 5‐azacytidine‐induced demethylation. Exp. Cell Res. 168: 153–159, 1987.
 74. Ferber, A., C. D. Chang, C. Sell, A. Ptasznik, V. J. Cristofalo, H. Ozer, D. Leroith, and R. Baserga. Failure of senescent human fibroblasts to express the IGF‐I gene. J. Biol. Chem. 268: 17883–17888, 1993.
 75. Frederich, K. M., P. D. Phillips, and V. J. Cristofalo. Stimulation of DNA synthesis in senescent human cells following incubation with plasma membranes. Exp. Cell Res. 202: 386–390, 1992.
 76. Friedman, D., and A. Globerson. Immune reactivity during aging. II. Analysis of the cellular mechanisms involved in the deficient antibody response in old mice. Mech. Ageing Dev. 7: 299–307, 1978.
 77. Fulder, S.J. The growth of cultured human fibroblasts treated with hydrocortisone and extracts of the medicinal plant Panax ginseng. Exp. Gerontol. 12: 125–131, 1977.
 78. Galloway, S. M., and K. E. Buckton. Aneuploidy and ageing: chromosome studies on a random sample of the population using G‐banding. Cytogenet. Cell Genet. 20: 78–95, 1978.
 79. Gerhard, G. S., P. D. Phillips, and V. J. Cristofalo. EGF‐and PDGF‐stimulated phosphorylation in young and senescent WI‐38 cells. Exp. Cell Res. 193: 87–92, 1991.
 80. Giordano, T., and D. N. Foster. Identification of a highly abundant cDNA isolated from senescent WI‐38 cells. Exp. Cell Res. 185: 399–406, 1989.
 81. Giordano, T., D. Kleinsek, and D. N. Foster. Increase in abundance of a transcript hybridizing to elongation factor I alpha during cellular senescence and quiescence. Exp. Gerontol. 24: 501–513, 1989.
 82. Goldstein, S. The role of DNA repair in aging of cultured fibroblasts from xeroderma pigmentosum and normals. Proc. Soc. Exp. Biol. Med. 137: 730–734, 1971.
 83. Goldstein, S., Senescence. In: Endocrinology, edited by L. J. Degroot, G. F. Cahill, Jr., W. D. Odell, L. Martini, J. T. Potts, Jr., D. H. Nelson, E. Steinberger, and A. I. Winegrad. New York: Grune and Stratton, 1979, vol. 3, p. 2001–2028.
 84. Goldstein, S. Replicative senescence: the human fibroblast comes of age. Science 249: 1129–1133, 1990.
 85. Goldstein, S., and D. P. Singal. Senescence of cultured human fibroblasts: mitotic vs. metabolic time. Exp. Cell Res. 88: 359–364, 1974.
 86. Goldstein, S., J. W. Littlefield, and J. S. Soeldner. Diabetes mellitus and aging: diminished plating efficiency of cultured human fibroblasts. Proc. Natl. Acad. Sci. USA 64: 155–160, 1969.
 87. Goldstein, S., E. J. Moerman, and R. C. Baxter. Accumulation of insulin‐like growth factor binding protein‐3 in conditioned medium of human fibroblasts increases with chronologic age of donor and senescence in vitro. J. Cell. Physiol. 156: 294–302, 1993.
 88. Goldstein, S., E. J. Moerman, R. A. Jones, and R. C. Baxter. Insulin‐like growth factor binding protein 3 accumulates to high levels in culture medium of senescent and quiescent human fibroblasts. Proc. Natl. Acad. Sci. USA 88: 9680–9684, 1991.
 89. Goldstein, S., E. J. Moerman, J. S. Soeldner, R. E. Gleason, and D. M. Barnett. Chronologic and physiologic age affect replicative life‐span of fibroblasts from diabetic, prediabetic, and normal donors. Science 199: 781–782, 1978.
 90. Gorman, S. D., and V. J. Cristofalo. Reinitiation of cellular DNA synthesis in BrdU‐selected nondividing senescent cells by simian virus 40 infection. J. Cell. Physiol. 125: 122–126, 1985.
 91. Gorman, S. D., and V. J. Cristofalo. Analysis of the G1 arrest position of senescent WI‐38 cells by quinacrine dihydrochloride nuclear fluorescence‐evidence for a late G1 arrest. Exp. Cell Res. 167: 87–94,1986.
 92. Gracy, R. W., K. U. Yuksel, M. L. Chapman, J. K. Cini, M. Jahani, H. S. Lu, B. Oray, and J. M. Talent. Impaired protein degradation may account for the accumulation of “abnormal” proteins in aging cells. In: Modern Aging Research, Modification of Proteins During Aging, edited by R. C. Adelman and E. E. Dekker. New York: Alan R. Liss, 1985, vol. 7, p. 1–18.
 93. Gray, M. D., S. A. Jesch, and G. H. Stein. 5‐Azacytidine‐induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts. J. Cell. Physiol. 149: 477–484, 1991.
 94. Greenberg, S. B., G. L. Grove, and V. J. Cristofalo. Cell size in aging monolayer cultures. In Vitro Cell. Dev. Biol. 13: 297–300, 1977.
 95. Grove, G. L., and V. J. Cristofalo. Characterization of the cell cycle of cultured human diploid cells: effects of aging and hydrocortisone. J. Cell. Physiol. 90: 415–422, 1977.
 96. Hadley, E. C., E. D. Kress, and V. J. Cristofalo. Trypsinization frequency and loss of proliferative capacity in WI‐38 cells. J. Gerontol. 34: 170–176, 1979.
 97. Haff, R. F., and H. E. Swim. Serial propagation of 3 strains of rabbit fibroblasts: their susceptibility to infection with vaccinia virus. Proc. Soc. Exp. Biol. Med. 93: 200–204, 1956.
 98. Hara, E., H. Tsurui, A. Shinozaki, S. Nakada, and K. Oda. Cooperative effect of antisense‐Rb and antisense‐p53 oligomers on the extension of lifespan in human diploid fibroblasts, TIG‐1. Biochem. Biophys. Res. Commun. 179: 528–534, 1991.
 99. Harley, C. B. Telomere loss: mitotic clock or genetic time bomb? Mutat. Res. 256: 271–282, 1991.
 100. Harley, C. B., and S. Goldstein. Cultured human fibroblasts: distribution of cell generations and a critical limit. J. Cell. Physiol. 97: 509–516, 1978.
 101. Harley, C. B., A. B. Futcher, and C. W. Greider. Telomeres shorten during aging of human fibroblasts. Nature 345: 458–460, 1990.
 102. Harley, C. B., H. Vaziri, C. M. Counter, and R. C. Allsopp. The telomere hypothesis of cellular aging. Exp. Gerontol. 27: 375–382, 1992.
 103. Harman, D. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11: 298–300, 1956.
 104. Harman, D., The free radical theory of aging. In: Modern Biological Theories of Aging, edited by H. R. Warner, R. N. Butler, R. L. Sprott, and E. L. Schneider. New York: Raven Press, 1987, p. 81–87.
 105. Harrison, D. E. Normal function of transplanted mouse erythrocyte precursors for 21 months beyond donor life spans. Nature New Biol. 237: 220–222, 1972.
 106. Harrison, D. E. Normal production of erythrocytes by mouse marrow continuous for 73 months. Proc. Natl. Acad. Sci. USA 70: 3184–3188, 1973.
 107. Harrison, D. E. Normal function of transplated marrow cell lines from aged mice. J. Gerontol. 30: 279–285, 1975.
 108. Harrison, D. E., and J. W. Doubleday. Normal function of immunologic stem cells from aged mice. J. Immunol. 114: 1314–1317, 1975.
 109. Harrison, D. E., C. M. Astle, and J. W. Doubleday. Cell lines from old immunodeficient donors give normal reponses in young recipients. J. Immunol. 118: 1223–1227, 1977.
 110. Hart, R. W., and R. B. Setlow. Correlation between deoxyribonucleic acid excision‐repair and lifespan in a number of mammalian species. Proc. Natl. Acad. Sci. USA 71: 2169–2173, 1974.
 111. Hart, R. W., and R. B. Setlow. DNA repair in late‐passage human cells. Mech. Ageing Dev. 5: 67–77, 1976.
 112. Hart, R. W., G. A. Sacher, and T. L. Hoskins. DNA repair in a short‐ and a long‐lived rodent species. J. Gerontol. 34: 808–817, 1979.
 113. Hauri, H.‐P., and A. Schweizer. The endoplasmic reticulum‐Golgi intermediate compartment. Curr. Opin. Cell Biol. 4: 600–608, 1992.
 114. Hay, R. J., Cell strain senescence in vitro: cell culture anomaly or an expression of a fundamental inability of normal cells to survive and proliferate. In: Aging in Cell and Tissue Culture, edited by E. Holeckova and V. J. Cristofalo. New York: Plenum Press, 1970, p. 7–24.
 115. Hay, R. J., and B. L. Strehler. The limited growth span of cell strains isolated from the chick embryo. Exp. Gerontol. 1: 123–135, 1967.
 116. Hay, R. J., R. A. Menzies, H. P. Morgan, and B. L. Strehler. The division potential of cells in continuous growth as compared to cells subcultivated after maintenance in stationary phase. Exp. Gerontol. 3: 35–44, 1968.
 117. Hayakawa, M. Progressive changes of the growth characteristics of the human diploid cells in serial cultivation in vitro. Tohoku J. Exp. Med. 98: 171–179, 1969.
 118. Hayflick, L. The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37: 614–636, 1965.
 119. Hayflick, L. Aging under glass. Exp. Gerontol. 5: 291–303, 1970.
 120. Hayflick, L., The cellular basis for biological aging. In: Handbook of the Biology of Aging, edited by C. E. Finch and L. Hayflick. New York: Van Nostrand Reinhold Company, 1977, p. 159–179.
 121. Hayflick, L., and P. S. Moorhead. The serial cultivation of human diploid cell strains. Exp. Cell Res. 25: 585–621, 1961.
 122. Hoehn, H., M. Simpson, E. M. Bryant, P. S. Rabinovitch, D. Salk, and G. M. Martin. Effects of chromosome constitution on growth and longevity of human skin fibroblast cultures. Am. J. Med. Genet. 7: 141–154, 1980.
 123. Holliday, R., and T. B. L. Kirkwood. Predictions of the somatic mutation and mortilization theories of cellular ageing are contrary to experimental observations. J. Theor. Biol. 93: 627–642, 1981.
 124. Holliday, R., and G. M. Tarrant. Altered enzymes in aging human fibroblasts. Nature 238: 26–30, 1972.
 125. Honda, S., and M. Matsuo. Relationship between the cellular glutathione level and in vitro life span of human diploid fibroblasts. Exp. Gerontol. 23: 81–86, 1988.
 126. Hosokawa, M., P. D. Phillips, and V. J. Cristofalo. The effect of dexamethasone on epidermal growth factor binding and stimulation of proliferation in young and senescent WI‐38 cells. Exp. Cell Res. 164: 408–414, 1986.
 127. Hunter, T. Braking the cycle. Cell 75: 839–841, 1993.
 128. Jensen, F., H. Koprowski, and J. A. Ponten. Rapid transformation of human fibroblast cultures by simian virus 40. Proc. Natl. Acad. Sci. USA 50: 343–348, 1963.
 129. Johnson, L. F., H. T. Abelson, S. Penman, and H. Green. The relative amounts of the cytoplasmic RNA species in normal, transformed, and senescent cultured cell lines. J. Cell. Physiol. 90: 465–470, 1976.
 130. Kaji, K., and M. Matsuo. A low density inoculation method for the serial subcultivation of human diploid fibroblasts: an efficient model for the study of cellular ageing. Mech. Ageing Dev. 13: 219–225, 1980.
 131. Klein, C. B., K. Conway, X. W. Wang, R. K. Bhamra, X. Lin, M. D. Cohen, L. Annab, J. C. Barrett, and M. Costa. Senescence of nickel‐transformed cells by an X chromosome: possible epigenetic control. Science 251: 796–799, 1991.
 132. Kleinsek, D. A. Selection of mRNAs expressed during cellular senescence in vitro. Age 12: 55–60, 1989.
 133. Krohn, P. L. Heterochronic transplantation in the study of ageing. In: Proceedings of the Royal Society, Review Lectures on Senescence, Series B, London: The Royal Society, 1962a, vol. 157, p. 128–147.
 134. Krohn, P. L., Transplantation and aging. In: Topics of the Biology of Aging, edited by P. L. Krohn. New York: J. Wiley, 1962b, p. 125–148.
 135. Krooth, R. S., M. W. Shaw, and B. K. Campbell. A persistent strain of diploid fibroblasts. J. Natl. Cancer Inst. 32: 1031–1040, 1964.
 136. Kumar, S., A. J. Millis, and C. Baglioni. Expression of interleukin 1‐inducible genes and production of interleukin 1 by aging human fibroblasts. Proc. Natl. Acad. Sci. USA 89: 4683–4687, 1992.
 137. Kumazaki, T., R. S. Robetorye, S. C. Robertorye, and J. R. Smith. Fibronectin expression increases during in vitro cellular senescence: correlation with increased cell area. Exp. Cell Res. 195: 13–19, 1991.
 138. Laishes, B. A., and G. M. Williams. Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology. In Vitro Cell. Dev. Biol. 12: 821–832, 1976.
 139. Lampidis, T. J., and G. E. Schraiberger. Age‐related loss of DNA repair synthesis in isolated rat myocardial cells. Exp. Cell Res. 96: 412–416, 1975.
 140. Leblond, C. P. Classification of cell populations on the basis of their proliferative behavior. Natl. Cancer Inst. Monogr. 14: 119–145, 1964.
 141. Le Guilly, Y., M. Simon, P. Lenoir, and M. Bourel. 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, 1973.
 142. Levy, J. A., M. Virolainen, and V. Defendi. Human lymphoblastoid lines from lymph node and spleen. Cancer Lett. 22: 517–524, 1968.
 143. Lipetz, J., and V. J. Cristofalo. Ultrastructural changes accompanying the aging of human diploid cells in culture. J. Ultrastruct. Res. 39: 43–56, 1972.
 144. Liu, A. Y. C., H. S. Choi, Y. K. Lee, and K. Y. Chen. Molecular events involved in transcriptional activation of heat shock genes become progressively refractory to heat stimulation during aging of human diploid fibroblasts. J. Cell. Physiol. 149: 560–566, 1991.
 145. Luce, M. C., and C. L. Bunn. Decreased accuracy of protein synthesis in extracts from aging human diploid fibroblasts. Exp. Gerontol. 24: 113–125, 1989.
 146. Luce, M. C., and V. J. Cristofalo. Reduction in heat shock gene expression correlates with increased thermosensitivity in senescent human fibroblasts. Exp. Cell Res. 202: 9–16, 1992.
 147. Lucibello, F. C., A. Sewing, S. Brusselbach, C. Burger, and R. Muller. Deregulation of cyclins D1 and E and suppression of cdk2 and cdk4 in senescent human fibroblasts. J. Cell Sci. 105: 123–133, 1993.
 148. Lumpkin, C. K. J., J. K. McClung, O. M. Pereira‐Smith, and J. R. Smith. Existence of high abundance antiproliferative mRNAs in senescent human diploid fibroblasts. Science 232: 393–395, 1986.
 149. Macieira‐Coelho, A. Action of cortisone on human fibroblasts in vitro. Experientia 22: 390–391, 1966.
 150. Macieira‐Coelho, A., The decreased growth potential in vitro of human fibroblasts of adult origin. In: Aging in Cell and Tissue Culture, edited by E. Holeckova and V. J. Cristofalo. New York: Plenum Press, 1970, p. 83–119.
 151. Macieira‐Coelho, A. Metabolism of aging cells in culture. Gerontology 22: 3–8, 1976.
 152. Macieira‐Coelho, A. Biology of Normal Proliferating Cells In Vitro. Relevance for In Vivo Aging. Basel: Karger, 1988.
 153. Macieira‐Coelho, A., and L. Berumen. The cell cycle during growth inhibition of human embryonic fibroblasts in vitro. Proc. Soc. Exp. Biol. Med. 144: 43–47, 1973.
 154. Macieira‐Coelho, A., and J. Ponten. Analogy in growth between late passage human embryonic and early passage human adult fibroblasts. J. Cell Biol. 43: 374–377, 1969.
 155. Macieira‐Coelho, A., J. Ponten, and L. Phillipson. The division cycle and RNA synthesis in diploid human cells at different passage levels in vitro. Exp. Cell Res. 42: 673–684, 1966.
 156. Mann, D. M., P. J. Mckeown‐Longo, and A. J. Millis. Binding of soluble fibronectin and its subsequent incorporation into the extracellular matrix by early and late passage human skin fibroblasts. J. Biol. Chem. 263: 2756–2760, 1988.
 157. Martin, G. M. Cellular aging: clonal senescence. Am. J. Pathol. 89: 484–511, 1977.
 158. Martin, G. M., Genetic syndromes in man with potential relevance to pathobiology of aging. In: Genetic Effects on Aging, edited by D. Bergsma and D. E. Harrison. New York: Alan R. Liss, 1978, p. 5–39.
 159. Martin, G. M., and C. A. Sprague. Clonal senescence and atherosclerosis. Lancet 2: 1370–1371, 1972.
 160. Martin, G. M., and C. A. Sprague. Symposium on in vitro studies related to atherogenesis. Life histories of hyperplastoid cell lines from aorta and skin. Exp. Mol. Pathol. 18: 125–141, 1973.
 161. Martin, G. M., C. E. Ogburn, and C. A. Sprague. Effects of age on cell division capacity. In: Aging: A Challenge to Science and Society, edited by D. Danon, N. W. Shock and M. Marois. Oxford: Oxford University Press, 1981, vol. 1, p. 124–135.
 162. Martin, G. M., C. A. Sprague, and C. J. Epstein. Replicative lifespan of cultivated human cells. Effects of donor age, tissue, and genotype. Lab. Invest. 23: 86–92, 1970.
 163. Martin, G. M., C. A. Sprague, T. H. Norwood, and W. R. Pendergrass. Clonal selection, attenuation and differentiation in an in vitro model of hyperplasia. Am. J. Pathol. 74: 137–154, 1974.
 164. Marx, J. How p53 suppresses cell growth. Science 262: 1644–1645, 1993.
 165. Matsumura, T. Multinucleation and polyploidization of aging human cells in culture. Adv. Exp. Med. Biol. 129: 31–38, 1980.
 166. Matsumura, T., Z. Zerrudo, and L. Hayflick. Senescent human diploid cells in culture: survival, DNA synthesis and morphology. J. Gerontol. 34: 328–334, 1979.
 167. Mattern, M. R., and P. A. Cerutti. Age‐dependent excision repair of damaged thymine from gamma‐irradiated DNA by isolated nuclei from human fibroblasts. Nature 254: 450–452, 1975.
 168. McHale, J. S., M. L. Mouton, and J. McHale. Limited culture lifespan of human diploid cells as a function of metabolic time instead of division potential. Exp. Gerontol. 6: 89–93, 1970.
 169. Meek, R. L., P. D. Bowman, and C. W. Daniel. Establishment of mouse embryo cells in vitro. Exp. Cell Res. 107: 277–284, 1977.
 170. Mellgren, J. Effects of the number of cell divisions and of added isologous nucleic acids on aging of normal human fibroblasts in vitro. Pathol. Eur. 10: 215–219, 1975.
 171. Merz, G. S., and J. D. Ross. Viability of human diploid cells as a function of in vitro age. J. Cell. Physiol. 74: 219–221, 1969.
 172. Millis, A. J., M. Hoyle, H. M. McCue, and H. Martini. Differential expression of metalloproteinase and tissue inhibitor of metalloproteinase genes in aged human fibroblasts. Exp. Cell Res. 201: 373–379, 1992.
 173. Millis, A. J., J. Sottile, M. Hoyle, D. M. Mann, and V. Diemer. Collagenase production by early and late passage cultures of human fibroblasts. Exp. Gerontol. 24: 559–575, 1989.
 174. Milo, G. E., and B. C. Castro. Conditions for transformation of human fibroblast cells: an overview. Cancer Lett. 31: 1–13, 1986.
 175. Mitsui, Y., and E. L. Schneider. Increased nuclear sizes in senescent human diploid fibroblast cultures. Exp. Cell Res. 100: 147–152, 1976a.
 176. Mitsui, Y., and E. L. Schneider. Relationship between cell replication and volume in senescent human diploid fibroblasts. Mech. Ageing Dev. 5: 45–56, 1976b.
 177. Mitsui, Y., J. R. Smith, and E. L. Schneider. Equivalent proliferation potential of different size classes of human diploid fibroblasts. J. Gerontol. 36: 416–419, 1981.
 178. Moore, G. E., and W. F. McLimans. The life span of the cultured normal cell: concepts derived from studies of human lymphoblasts. J. Theor. Biol. 20: 217–226, 1968.
 179. Morley, A. A. Is ageing the result of dominant or co‐dominant mutations? J. Theor. Biol. 98: 469–474, 1982.
 180. Morris, C. M., A. E. Reeve, P. H. Fitzgerald, P. E. Hollings, M. E. J. Beard, and D. C. Heaton. Genomic diversity correlates with clinical variation in Ph‐negative chronic myeloid leukaemia. Nature 320: 281–283, 1986.
 181. Mueller, S. N., E. M. Rosen, and E. M. Levine. Cellular senescence in a cloned strain of bovine fetal aortic endothelial cells. Science 207: 889–891, 1980.
 182. Murano, S., R. Thweatt, R. J. Shmookler‐Reis, R. A. Jones, E. J. Moerman, and S. Goldstein. Diverse gene sequences are overexpressed in Werner syndrome fibroblasts undergoing premature replicative senescence. Mol. Cell. Biol. 11: 3905–3914,1991.
 183. Murato, S.‐I., Y. Mitsui, and M. Kawamura. Effect of in vitro aging on 6‐ketoprostaglandin F1α‐producing activity in cultured human diploid lung fibroblasts. Biochim. Biophys. Acta 574: 351–355, 1979.
 184. Nasmyth, K., and T. Hunt. Dams and sluices. Nature 366: 634–635, 1993.
 185. Nielsen, P. J., and J. M. Ryan. Cumulative population doublings as the determinant of chick cell lifespan in vitro. J. Cell. Physiol. 107: 371–378, 1981.
 186. Nienhaus, A. J., B. De Jong, L. P. Ten Kate, and F. H. Oswald. Fibroblast culture in Werner's syndrome. Humattgenetik 13: 244–246, 1971.
 187. Ning, Y., J. C. Weber, A. M. Killary, D. H. Ledbetter, J. R. Smith, and O. M. Pereira‐Smith. Genetic analysis of indefinite division in human cells: evidence for a cell senescence‐related gene(s) on human chromosome 4. Proc. Natl. Acad. Sci. USA 88: 5635–5639, 1991.
 188. Noda, A., Y. Ning, S. F. Venable, O. M. Pereira‐Smith, and J. R. Smith. Cloning of senescent cell derived inhibitors of DNA synthesis using an expression screen. Exp. Cell Res. 211: 90–98, 1994.
 189. Norwood, T. H., W. R. Pendergrass, C. A. Sprague, and G. M. Martin. Dominance of the senescent phenotype in heterokaryons between replicative and post‐replicative human fibroblast‐like cells. Proc. Natl. Acad. Sci. USA 71: 2231–2235, 1974.
 190. Norwood, T. H., A. Saulewicz, F. Hanaoka, and W. R. Pendergrass. Failure by senescent fibroblasts to complement a DNA polymerase a mutant (Abstract). Ann. Meet. Geroniol. Soc. Am. 44th San Francisco CA 1991, p. 354. Published in The Gerontologist by the Gerontological Society of America (GSA).
 191. Norwood, T. H., J. R. Smith, and G. H. Stein. Aging at the cellular level: the human fibroblastlike cell model. In: Handbook of the Biology of Aging, edited by E. L. Schneider and J. W. Rowe. San Diego: Academic Press, 1990, p. 131–154.
 192. Ogden, D. A., and H. S. Micklem. The fate of serially transplanted bone marrow cell populations from young and old donors. Transplantation 22: 287–293, 1976.
 193. Olashaw, N. E., E. D. Kress, and V. J. Cristofalo. Thymidine triphosphate synthesis in senescent WI38 cells. Relationship to loss of replicative capacity. Exp. Cell Res. 149: 547–554, 1983.
 194. Olsson, L., and P. Ebbesen. Aging decreases the activity of epidermal G1 and G2 inhibitors in mouse skin independent of grafting on old or young recipients. Exp. Gerontol. 12: 59–62, 1977.
 195. Orgel, L. E. The maintenance of the accuracy of protein synthesis and its relevance to aging. Proc. Natl. Acad. Sci. USA 49: 517–521, 1963.
 196. Painter, R. B., J. M. Clarkson, and B. R. Young. Ultraviolet‐induced repair replication in aging diploid human cells (WI‐38). Radiat. Res. 56: 560–564, 1973.
 197. Pappenholz, V. Correlation between DNA repair of embryonic fibroblasts and different life span of 3 inbred mouse strains. Mech. Ageing Dev. 7: 131–150, 1978.
 198. Paulsson, Y., M. Bywater, S. Pfeifer‐Ohlsson, R. Ohlsson, S. Nilsson, C. H. Heldin, B. Westermark, and C. Betsholtz. Growth factors induce early pre‐replicative changes in senescent human fibroblasts. EMBO J. 5: 2157–2162, 1986.
 199. Pendergrass, W. R., J. C. Angello, M. D. Kirschner, and T. H. Norwood. 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, 1991a.
 200. Pendergrass, W. R., J. C. Angello, A. C. Saulewicz, and T. H. Norwood. DNA polymerase alpha and the regulation of entry into S phase in heterokaryons. Exp. Cell Res. 192: 426–432, 1991b.
 201. Pendergrass, W. R., G. M. Martin, and P. Bornstein. Evidence contrary to the protein error hypothesis for in vitro senescence. J. Cell. Physiol. 87: 3–13, 1976.
 202. Pereira‐Smith, O. M., and J. R. Smith. Phenotype of low proliferative potential is dominant in hybrids of normal human fibroblasts. Somatic Cell Mol. Genet. 8: 731–742, 1982.
 203. Pereira‐Smith, O. M., and J. R. Smith. Evidence for the recessive nature of cellular immortality. Science 221: 964–966, 1983.
 204. Pereira‐Smith, O. M., and J. R. Smith. Genetic analysis of indefinite division in human cells: identification of four complementation groups. Proc. Natl. Acad. Sci. USA 85: 6042–6046, 1988.
 205. Pereira‐Smith, O. M., S. F. Fisher, and J. R. Smith. Senescent and quiescent cell inhibitors of DNA synthesis. Membrane‐associated proteins. Exp. Cell Res. 160: 297–306, 1985.
 206. Peterson, C., and J. E. Goldman. Alterations in calcium content and biochemical processes in cultured skin fibroblasts from aged and Alzheimer donors. Proc. Natl. Acad. Sci. USA 83: 2758–2762, 1986.
 207. Peterson, C., R. R. Ratan, M. L. Shelanski, and J. E. Goldman. Cytosolic free calcium and cell spreading decrease in fibroblasts from aged and Alzheimer donors. Proc. Natl. Acad. Sci. USA 83: 7999–8001, 1986.
 208. Petursson, G., J. G. Coughlin, and C. Meylan. Long‐term cultivation of diploid rat cells. Exp. Cell Res. 33: 60–67, 1964.
 209. Phillips, P. D., K. Kaji, and V. J. Cristofalo. Progressive loss of the response of senescing WI‐38 cells to platelet‐derived growth factor, epidermal growth factor, insulin, transferrin, and dexamethasone. J. Gerontol. 39: 11–17, 1984.
 210. Phillips, P. D., E. Kuhnle, and V. J. Cristofalo. [125I] EGF binding ability is stable throughout the replicative life span of WI‐38 cells. J. Cell. Physiol. 114: 311–316, 1983.
 211. Phillips, P. D., R. J. Pignolo, and V. J. Cristofalo. Insulinlike 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, 1987.
 212. Phillips, P. D., R. J. Pignolo, K. Nishikura, and V. J. Cristofalo. Renewed DNA synthesis in senescent WI‐38 cells by expression of an inducible chimeric c‐fos construct. J. Cell. Physiol. 151: 206–212, 1992.
 213. Pignolo, R. J., E. J. Masoro, W. W. Nichols, C. I. Bradt, and V. J. Cristofalo. Skin fibroblasts from Fischer 344 rats undergo similar changes in replicative life span but not immortalization with caloric restriction of donors. Exp. Cell Res. 201: 16–22, 1992.
 214. Pignolo, R. J., M. O. Rotenberg, and V. J. Cristofalo. Differential expression of a novel cell cycle‐regulated transcript in late population doubling level WI‐38 cells (Abstract). Ann. Meet. Gerontol. Soc. Am. 44th San Francisco CA 1991, p. 74. published in The Gerontologist by Gerontological Society of America (GSA).
 215. Pignolo, R. J., M. O. Rotenberg, and V. J. Cristofalo. Senescent WI‐38 cells fail to express EPC‐1, a gene induced in young cells upon entry into the Go state. J. Biol. Chem. 268: 8949–8957, 1993.
 216. Pignolo, R. J., M. O. Rotenberg, and V. J. Cristofalo. Alterations in contact and density‐dependent arrest state in senescent WI‐38 cells. In Vitro Cell. Dev. Biol. 30: 471–476, 1994.
 217. Pious, D. A., R. N. Hamburger, and S. E. Mills. Clonal growth of primary human cell cultures. Exp. Cell Res. 33: 495–507, 1964.
 218. Plisko, A., and B. A. Gilchrest. Growth factor responsiveness of cultured human fibroblasts declines with age. J. Gerontol. 38: 513–518, 1983.
 219. Pochron, S. F., A. R. O'Meara, and M. J. Kurtz. Control of transcription in aging WI‐38 cells stimulated by serum to divide. Exp. Cell Res. 116: 63–74, 1978.
 220. Ponten, J., Aging properties of glia. In: INSERM, edited by F. Bourliere, Y. Courtois, A. Macieira‐Coelho and L. Robert. Paris: INSERM, 1973, vol. 27, p. 53–64.
 221. Porter, M. B., O. M. Pereira‐Smith, and J. R. Smith. Novel monoclonal antibodies identify antigenic determinants unique to cellular senescence. J. Cell. Physiol. 142: 425–433, 1990.
 222. Praeger, F. C., and V. J. Cristofalo. The growth of WI‐38 cells in a serum‐free, growth factor‐free, medium with elevated calcium concentrations. In Vitro Cell. Dev. Biol. 22: 355–359, 1986a.
 223. Praeger, F. C., and V. J. Cristofalo. Modulation of WI‐38 cell proliferation by elevated levels of CaCl2. J. Cell. Physiol. 129: 27–35, 1986b.
 224. Praeger, F. C., and B. A. Gilchrest. Influence of increased extracellular calcium concentration and donor age on density‐dependent growth inhibition of human fibroblasts. Proc. Soc. Exp. Biol. Med. 182: 315–321, 1986.
 225. Price, G. B., and T. Makinodan. Immunologic deficiencies in senescence. II. Characterization of extrinsic deficiencies. J. Immunol. 108: 413–417, 1972.
 226. Puck, T. T., C. A. Waldren, and J. H. Tjio. Some data bearing on the long term growth of mammalian cells. In: Symposium on Topics in the Biology of Aging, edited by P. L. Krohn. New York: J. Wiley, 1966, p. 101–123.
 227. Quinn, L. S., T. H. Norwood, and M. Nameroff. Myogenic stem cell commitment probability remains constant as a function of organismal and mitotic age. J. Cell. Physiol. 134: 324–336, 1988.
 228. Radna, R. L., Y. Caton, K. K. Jha, P. Kaplan, G. Li, F. Tragonos, and H. L. Ozer. Immortalization of origin‐defective simian virus 40 tsA‐transformed human fibroblasts is temperature dependent. Mol. Cell. Biol. 9: 3093–3096, 1989.
 229. Ransom, R. Computers and Embryos: Models in Developmental Biology. New York: J. Wiley, 1981.
 230. Regan, J. D., M. M. Sigel, W. H. Lee, K. A. Llamas, and A. R. Beasley. Chromosomal alterations in marine fish cells in vitro. Can. J. Genet. Cytol. 10: 448–453, 1968.
 231. Reznick, A. Z., A. Dovrat, L. Rosenfelder, S. Shupund, and D. Gershon. Defective enzyme molecules in cells of aging animals are partially denatured, totally inactive, normal degradation intermediates. In: Modern Aging Research, Modification of Proteins During Aging, edited by R. C. Adelman and E. E. Dekker. New York: Alan R. Liss, 1985, vol. 7, p. 69–81.
 232. Rheinwald, J. G., and H. Green. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331–334, 1975.
 233. Riabowol, K., J. Schiff, and M. Z. Gilman. Transcription factor AP‐1 activity is required for initiation of DNA synthesis and is lost during cellular aging. Proc. Natl. Acad. Sci. USA 89: 157–161, 1992.
 234. Rittling, S. R., K. M. Brooks, V. J. Cristofalo, and R. Baserga. Expression of cell cycle‐dependent genes in young and senescent WI‐38 fibroblasts. Proc. Natl. Acad. Sci. USA 83: 3316–3320, 1986.
 235. Robbins, E., E. M. Levine, and H. Eagle. Morphological changes accompanying senescence of cultured human diploid cells. J. Exp. Med. 131: 1211–1222, 1970.
 236. Róhme, D. 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, 1981.
 237. Rose, D. W., G. McCabe, J. R. Feramisco, and M. Adler. Expression of c‐fos and AP‐1 activity in senescent human fibroblasts is not sufficient for DNA synthesis. J. Cell. Biol. 119: 1405–1411, 1992.
 238. Rosendaal, M., G. S. Hodgson, and T. R. Bradley. Haemopoietic stem cells are organised for use on the basis of their generation age. Nature 264: 68–69, 1976.
 239. Rothstein, M., The alteration of enzymes in aging. In: Modern Aging Research, Modification of Proteins During Aging, edited by R. C. Adelman and E. E. Dekker. New York: Alan R. Liss, 1985, vol. 7, p. 53–67.
 240. Rowe, D. W., B. J. Starman, W. Y. Fujimoto, and R. H. Williams. Differences in growth response to hydrocortisone and ascorbic acid by human diploid fibroblasts. In Vitro Cell. Dev. Biol. 13: 824–830, 1977.
 241. Ryan, J. M., and V. J. Cristofalo. Chromatin template activity during aging in WI‐38 cells. Exp. Cell Res. 90: 456–458, 1975.
 242. Ryan, J. M., G. Duda, and V. J. Cristofalo. Error accumulation and aging in human diploid cells. J. Gerontol. 19: 616–621, 1974.
 243. Ryan, J. M., D. G. Ostrow, X. O. Breakefield, E. S. Gershon, and L. Upchurch. A comparison of the proliferative and replication lifespan kinetics of cell cultures derived from monozygotic twins. In Vitro Cell. Dev. Biol. 17: 20–27, 1981.
 244. Ryan, P. A., V. M. Maher, and J. J. McCormick. Failure of infinite life span human cells from different immortality complementation groups to yield finite life span hybrids. J. Cell. Physiol. 159: 151–160, 1994.
 245. Schneider, E. L., and C. J. Epstein. Replication rate and lifespan of cultured fibroblasts in Down's syndrome. Proc. Soc. Exp. Biol. Med. 141: 1092–1094, 1972.
 246. Schneider, E. L., and B. J. Fowlkes. Measurement of DNA content and cell volume in senescent human fibroblasts utilizing flow multiparameter single cell analysis. Exp. Cell Res. 98: 298–302, 1976.
 247. Schneider, E. L., and Y. Mitsui. The relationship between in vitro cellular aging and in vivo human age. Proc. Natl. Acad. Sci. USA 73: 3548–3588, 1976.
 248. Schneider, E. L., and S. S. Shorr. Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts. Cell 6: 179–184, 1975.
 249. Schneider, E. L., Y. Mitsui, R. Tice, S. S. Shorr, and K. Braunschweiger. Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts. II. Synthesis and processing of RNA. Mech. Ageing Dev. 4: 449–458, 1975.
 250. Schwartz, A. G. Correlation between species life span and capacity to activate 7,12‐dimethylbenz (a) anthracene to a form mutagenic to a mammalian cell. Exp. Cell Res. 94: 445–447, 1975.
 251. Sell, C., A. Ptasznik, C. D. Chang, J. Swantek, V. J. Cristofalo, and R. Baserga. IGF‐I receptor levels and the proliferation of young and senescent human fibroblasts. Biochem. Biophys. Res. Commun. 194: 259–265, 1993.
 252. Seshadri, T., and J. Campisi. Repression of c‐fos transcription and an altered genetic program in senescent human fibroblasts. Science 247: 205–209, 1990.
 253. Seshadri, T., J. A. Uzman, J. Oshima, and J. Campisi. Identification of a transcript that is down‐regulated in senescent human fibroblasts. J. Biol. Chem. 268: 18474–18480, 1993.
 254. Shay, J. W., O. M. Pereira‐Smith, and W. E. Wright. A role for both RB and p53 in the regulation of human cellular senescence. Exp. Cell Res. 196: 33–39, 1991.
 255. Shay, J. W., M. D. West, and W. E. Wright. Re‐expression of senescent markers in deinduced reversibly immortalized cells. Exp. Gerontol. 27: 477–492, 1992.
 256. Shay, J. W., W. R. Wright, and H. Werbin. Defining the molecular mechanisms of human cell immortalization. Biochim. Biophys. Acta 1072: 1–7, 1991.
 257. Shigeoka, H., and H. C. Yang. Early kinase C dependent events in aging human diploid fibroblasts. Mech. Ageing Dev. 55: 49–59, 1990.
 258. Siminovitch, L., J. E. Till, and E. A. McCulloch. Decline in colony‐forming ability of marrow cells subjected to serial transplantation into irradiated mice. J. Cell. Physiol. 64: 23–31, 1964.
 259. Simons, J. W. J. W., A theoretical and experimental approach to the relationship between cell variability and aging in vitro. In: Aging in Cell and Tissue Culture, edited by E. Holeckova and V. J. Cristofalo. New York: Plenum Press, 1970, p. 25–39.
 260. Simons, J. W. I. M., and C. van den Broek. Comparison of ageing in vitro and ageing in vivo by means of size analysis using a Coulter counter. Gerontologia 16: 340–351, 1970.
 261. Slayback, J. R. B., L. W. Y. Cheung, and R. P. Geger. 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, 1977.
 262. Smith, B. T., J. S. Torday, and C. J. P. Giroud. The growth promoting effect of Cortisol in human fetal lung cells. Steroids 22: 515–524, 1973.
 263. Smith, C. A., and P. C. Hanawalt. Repair replication in cultured normal and transformed human fibroblasts. Biochim. Biophys. Acta 447: 121–132, 1976.
 264. Smith, J. R. Inhibitors of DNA synthesis derived from senescent human diploid fibroblasts. Exp. Gerontol. 27: 409–412, 1992.
 265. Smith, J. R., and L. Hayflick. Variation in the life‐span of clones derived from human diploid cell strains. J. Cell Biol. 62: 48–53, 1974.
 266. Smith, J. R., and R. G. Whitney. Intraclonal variation in proliferative potential of human diploid fibroblasts: stochastic mechanism for cellular aging. Science 207: 82–84, 1980.
 267. Smith, J. R., O. Pereira‐Smith, and E. L. Schneider. Colony size distribution as a measure of in vivo and in vitro aging. Proc. Natl. Acad. Sci. USA 75: 1353–1356, 1978.
 268. Sottile, J., M. Hoyle, and A. J. Millis. Differential response of early and late passage fibroblasts to collagenase stimulatory factor in conditioned media. Coll. Relat. Res. 8: 361–374, 1988.
 269. Sottile, J., D. M. Mann, V. Diemer, and A. J. Millis. Regulation of collagenase and collagenase mRNA production in early‐ and late‐passage human diploid fibroblasts. J. Cell. Physiol. 138: 281–290, 1989.
 270. Soukupova, M., E. Holeckova, and P. Hnevkovsky. Changes of the latent period of explanted tissues during ontogenesis. In: Aging in Cell and Tissue Culture, edited by E. Holeckova and V. J. Cristofalo. New York: Plenum Press, 1970, p. 41–56.
 271. Stanley, J., D. Pye, and A. Macgregor. Comparison of doubling numbers attained by cultured animal cells with life span of species. Nature 255: 158–159, 1975.
 272. Stanulis‐Praeger, B. M. Cellular senescence revisited: a review. Mech. Ageing Dev. 38: 1–48, 1987.
 273. Stein, G. H. SV40‐transformed human fibroblasts: evidence for cellular aging in precrisis cells. J. Cell. Physiol. 125: 36–44, 1985.
 274. Stein, G. H., and L. Atkins. Membrane‐associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: characterization and comparison to quiescent cell inhibitor. Proc. Natl. Acad. Sci. USA 83: 9030–9034, 1986.
 275. Stein, G. H., and R. M. Yanishevsky. Entry into S phase is inhibited in two immortal cell lines fused to senescent human diploid cells. Exp. Cell Res. 120: 155–165, 1979.
 276. Stein, G. H., and R. M. Yanishevsky. Quiescent human diploid cells can inhibit entry into S phase in replicative nuclei in heterodikaryons. Proc. Natl. Acad. Sci. USA 78: 3025–3029, 1981.
 277. Stein, G. H., R. M. Yanishevsky, L. Gordon, and M. Beeson. Carcinogen‐transformed human cells are inhibited from entry into S phase by fusion to senescent cells but cells transformed by DNA tumor viruses overcome the inhibition. Proc. Natl. Acad. Sci. USA 79: 5287–5291, 1982.
 278. Stein, G. H., L. Atkins, M. Beeson, and L. Gordon. Quiescent human diploid fibroblasts: common mechanism for inhibition of DNA replication in density‐inhibited and serum‐deprived cells. Exp. Cell Res. 162: 255–260, 1986.
 279. Stein, G. H., M. Beeson, and L. Gordon. Failure to phosphorylate retinoblastoma gene product in senescent human fibroblasts. Science 249: 666–669, 1990.
 280. Stein, G. H., L. F. Drullinger, R. S. Robetorye, O. M. Pereira‐Smith, and J. R. Smith. Senescent cells fail to express cdc2, cyc A, and cycB in response to mitogen stimulation. Proc. Natl. Acad. Sci. USA 88: 11012–11016, 1991.
 281. Steinhagen, M. Effect of donor age on clonal differentiation of human skin fibroblasts in vitro. Gerontology 31: 27–38, 1985.
 282. Suda, Y., M. Suzuki, Y. Ikawa, and S. Aizawa. Mouse embryonic stem cells exhibit indefinite proliferative potential. J. Cell. Physiol. 33: 197–201, 1987.
 283. Sugawara, O., M. Oshimura, M. Koi, L. A. Annab, and J. C. Barrett. Induction of cellular senescence in immortalized cells by human chromosome 1. Science 247: 707–710, 1990.
 284. Swim, H. E., and R. F. Parker. Culture characteristics of human fibroblasts propagated serially. Am. J. Hyg. 66: 235, 1957.
 285. Szilard, L. On the nature of the aging process. Proc. Natl. Acad. Sci. USA 45: 30–45, 1959.
 286. Tassin, J., E. Malaise, and Y. Courtois. Human lens cells have an in vitro proliferative capacity inversely proportional to the donor age. Exp. Cell Res. 123: 388–392, 1979.
 287. Thrash, C. R., and D. D. Cunningham. Stimulation of division of density‐inhibited fibroblasts by glucocorticoids. Nature 242: 399–401, 1973.
 288. Thweatt, R., C. K. Lumpkin, and S. Goldstein. A novel gene encoding a smooth muscle protein is overexpressed in senescent human fibroblasts. Biochem. Biophys. Res. Commun. 187: 1–7, 1992.
 289. Thweatt, R., S. Murano, R. D. Fleischmann, and S. Goldstein. Isolation and characterization of gene sequences over‐expressed in Werner syndrome fibroblasts during premature replicative senescence. Exp. Gerontol. 27: 433–440, 1992.
 290. Tice, R. R., E. L. Schneider, D. Kram, and P. Thorne. Cytokinetic analysis of impaired proliferative response of peripheral lymphocytes from aged humans to phytohemagglutinin. J. Exp. Med. 149: 1029–1041, 1979.
 291. Todaro, G. J., and H. Green. Serum albumin supplemented medium for long‐term cultivation of mammalian fibroblast strains. Proc. Soc. Exp. Biol. Med. 116: 688–692, 1964.
 292. Todaro, G. J., S. R. Wolman, and H. Green. Rapid transformation of human fibroblasts with low growth potential into established cell lines by SV40. J. Cell. Physiol. 62: 257–265, 1963.
 293. Valenti, C., and E. A. Friedman. Long‐term cultivation of diploid rabbit skin cells. Tex. Rep. Biol. Med. 26: 363–380, 1968.
 294. Vincent, R. A., and P. C. Huang. The proportion of cells labeled with tritiated thymidine as a function of population doubling level in cultures of fetal, adult, mutant, and tumor origin. Exp. Cell Res. 102: 31–42, 1976.
 295. Wang, E. A 57,000‐mol‐wt. protein uniquely present in non‐proliferating cells and senescent human fibroblasts. J. Cell Biol. 100: 545–551, 1985a.
 296. Wang, E. Rapid disappearance of statin, a nonproliferating and senescent‐cell specific protein, upon reentering the process of cell cycling. J. Cell Biol. 101: 1695–1701, 1985b.
 297. Wang, E. Statin, a non‐proliferation‐specific protein, is associated with the nuclear envelope and is heterogeneously distributed in cells leaving quiescent state. J. Cell. Physiol. 140: 418–426, 1989.
 298. Wang, E., and S. L. Lin. Disappearance of statin, a protein marker for nonproliferating and senescent cells, following serum‐stimulated cell‐cycle entry. Exp. Cell Res. 167: 135–143, 1986.
 299. Wang, E., and G. Tomaszewski. Granular presence of terminin is the marker to distinguish between the senescent and quiescent states. J. Cell. Physiol. 147: 514–522, 1991.
 300. Weissman‐Shomer, P., and M. Fry. Chick embryo fibroblast senescence in vitro: pattern of cell division and life span as a function of cell density. Mech. Ageing Dev. 4: 159–166, 1975.
 301. West, M. D., W. E. Wright, and J. W. Shay. Transcriptional mechanisms regulating the over‐expression of plasminogen activator inhibitor‐1 in senescent fibroblasts (Abstract). Ann. Meet. Gerontol. Soc. Am. 44th San Francisco CA 1991, p. 314. published in The Gerontologist by the Gerontological Society of America (GSA).
 302. Whitaker, N. J., E. L. Kidston, and R. R. Reddel. Finite life span of hybrids formed by fusion of different simian virus 40‐immortalized human cell lines. J. Virol. 66: 1202–1206, 1992.
 303. Williams, J. R., and K. L. Dearfield. Nonhuman fibroblast‐like cells in culture. In: CRC Handbook of Cell Biology of Aging, edited by V. J. Cristofalo. Boca Raton, FL: CRC Press, 1985, p. 433–451.
 304. Willie, J. J., N. R. Pittelkow, G. D. Shipley, and R. E. Scott. Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum‐free medium: clonal analyses, growth kinetics and cell cycle studies. J. Cell. Physiol. 121: 31–44, 1984.
 305. Wistrom, C., and B. Villeponteau. Cloning and expression of SAG: a novel marker of cellular senescence. Exp. Cell Res. 199: 355–362, 1992.
 306. Won, K.‐A., Y. Xiong, D. Beach, and M. Z. Gilman. Growth‐regulated expression of D‐type cyclin genes in human diploid fibroblasts. Proc. Natl. Acad. Sci. USA 89: 9910–9914, 1992.
 307. Wright, W. E., and J. W. Shay. Telomere positional effects and the regulation of cellular senescence. Trends Genet. 8: 193–197, 1992.
 308. Wright, W. E., O. M. Pereira‐Smith, and J. W. Shay. Reversible cellular senescence: implications for immortalization of normal human diploid cells. Mol. Cell. Biol. 9: 3088–3092, 1989.
 309. Yaffe, D. Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. USA 61: 477–483, 1968.
 310. Yanishevsky, R., and A. V. Carrano. Prematurely condensed chromosomes of dividing and non‐dividing cells in aging human cell cultures. Exp. Cell Res. 90: 169–174, 1975.
 311. Yanishevsky, R. M., and G. H. Stein. Ongoing DNA synthesis continues in young human diploid cells (HDC) fused to senescent HDC, but entry into S phase is inhibited. Exp. Cell Res. 126: 469–472, 1980.
 312. Yanishevsky, R., M. L. Mendelsohn, B. H. Mayall, and V. J. Cristofalo. Proliferative capacity and DNA content of aging human diploid cells in culture: a cytophotometric and autoradiographic analysis. J. Cell. Physiol. 84: 165–170, 1974.

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Vincent J. Cristofalo, Robert J. Pignolo. Cell Culture as a Model. Compr Physiol 2011, Supplement 28: Handbook of Physiology, Aging: 53-82. First published in print 1995. doi: 10.1002/cphy.cp110104