Cell Fusion Studies and Biochemical Analysis of DNA Synthesis in Werner and Non-Werner Cultured Cells

  • William Pendergrass
  • Darrell Salk
  • Thomas Norwood
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 190)


Cell fusion has been used extensively to determine whether DNA synthesis can be reinitiated (“rescued”) in senescent human diploid fibroblast-like (HDFL) cells. These experiments have shown that temporary resumption of DNA synthesis can occur after fusion of senescent cells to some, but not all, continuously propagated cell lines. In contrast, fusion of senescent HDFL cells to early passage HDFL cells, never results in rescue. In this monograph, the literature describing these experiments is reviewed. In addition, we propose a model which predicts that DNA synthesis is initiated in heterokaryons between senescent HDFL cells and other cell types only when the proliferating parental cell types donate enough DNA replication factors to the common pool to exceed the threshold concentration required for initiation of DNA synthesis. Evidence is presented indicating that senescent HDFL cells cultured from patients with Werner syndrome (referred to here as Werner cells) possess an increased requirement for these replication factors. However, we will describe studies indicating that DNA polymerase alpha remains inducible following mitogen stimulation senescent cultures derived from both normal and Werner donors. Thus, senescent HDFL cells may be in a metabolic state distinct from other quiescent cell types.


Senescent Cell T98G Cell Replication Factor Werner Syndrome Thymidine Label Index 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aso, K., Kondo, S., and Amano, M., 1980, A case of Werner’s syndrome in which lowered activity of platelet-dependent serum factor for cell growth and reduced growth potential of fibroblasts and epidermal cells were demonstrated, Nippon Hifuka Gakka Zasshi, 90: 929.Google Scholar
  2. Baril, E., Baril, B., Elford, H., and Luftig, R.B., 1974, DNA polymerase and a possible multi-enzyme complex for DNA biosynthesis eukaryotes, in: “Mechanism and Regulation of DNA Replication,” A. Kolber and K. Kohigam eds. Plenum Publishing Corp., New York, pp. 276–293.Google Scholar
  3. Bunn, C.L. and Tarrant, G.M., 1980, Limited lifespan in somatic cell hybrids and cybrids, Exp. Cell Res., 127: 385.Google Scholar
  4. Burmer, G.C., Motulsky, H., Zeigler, C.J., and Norwood, T.H.,1983, Inhibition of DNA synthesis in young cycling human diploid fibroblast-like cells upon fusion to enucleate cytoplasms from senescent cells, Exp. Cell Res., 145: 79.Google Scholar
  5. Burmer, G.C., Zeigler, C.J., and Norwood, T.H., 1982, Evidence for endogenous polypeptide-mediate inhibition of cell-cycle transit in human diploid, J. Cell Biol., 94: 187.PubMedCrossRefGoogle Scholar
  6. Croce, C.M. and Koprowski, H., 1974, Positive control of transformed phenotype in hybrid between SV-40 and normal human cells, Science, 184: 1288.PubMedCrossRefGoogle Scholar
  7. Drescher-Lincoln, C.K. and Smith, J.L., 1983, Inhibition of DNA synthesis in proliferating human diploid fibroblasts by fusion with senescent cytoplasts, Exp. Cell. Res., 144: 445.Google Scholar
  8. DePamphilis, M.L. and Wassarman, M.L., 1980, Replication of eukaryotic chromosomes: A close-up of the replication fork, Annu. Rev. Biochem., 49: 627.Google Scholar
  9. Duthu, G.S., Braunschweiger, K.I., Pereira-Smith, 0.M., Norwood, T.H., and Smith, J.R., 1982, A long-lived human diploid fibroblast line for cellular aging studies: applications in cell hybridization, Mech. Ageing Devel. 20: 243.Google Scholar
  10. Fialo, M. and Kenny, G.E., 1966, Enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesuim and calcium, J. Bacteriol. 92: 1710.Google Scholar
  11. Fry, M., 1983, in: “Eukaryotic DNA Polymerases,” L.S. Hnilica, ed., CRC Press Series in Biochemistry and Molecular Biology of the Cell Nucleus, CRC press.Google Scholar
  12. Gopalakrishman, T.V. and Anderson, W.F., 1979, Epigenetic activation of phenylalinine in mouse erythroleukemia cells by the cytoplast of rat hepatoma cells, Proc. Natl. Acad. Sci., 76: 3922.Google Scholar
  13. Goldstein, S., 1979, Studies on age-related diseases in cultured skin fibroblasts. J. Invest. Dermatol., 73: 19.Google Scholar
  14. Goldstein, S. and Lin, C.C., 1972, Rescue of senescent human fibroblasts by hybridization with hamster cells in vitro, Exp. Cell. Res., 70: 436.Google Scholar
  15. Hoehn, H., Bryant, E.M., Johnston, P., Norwood, T.H. and Martin, G.M., 1975, Non-selective isolation, stability and longevity of hybrids between normal human somatic cells, Nature 258: 608.PubMedCrossRefGoogle Scholar
  16. Hoehn, H., Bryant, E.M. and Martin, G.M., 1978, The replicative life spans of euploid hybrids derived from short-lived and long-lived human skin fibroblast cultures, Cytogenet. Cell Genet., 21: 282.Google Scholar
  17. Holm, C., 1982, Clonal lethality caused by the yeast plasmid 2’DNA, Cell, 29: 585.PubMedCrossRefGoogle Scholar
  18. Johnston, L.H. and Nasmyth, K.A., 1978, Saccharomyces Cerevisiae cell cycle mutant cdc 9 is defective in DNA, Lyare. Nat., 274: 891.Google Scholar
  19. Kahn, C.R., Gopalakrishman, T.V., and Weiss,“M.C., 1981, Transfer of heritable propérties by cell cybridization: Specificity and the role of selective pressure, Somat. Cell. Genet., 7: 547.Google Scholar
  20. Kornberg, A., 1980, Eukaryotic DNA polymerases, in: “DNA Replication,” A. Kornburg, ed., W.H. Freeman and Co., San Francisco, pp. 210–229.Google Scholar
  21. Lipsich, L.A., Kates, J.R., and Lucas, J.J., 1979, Expression of a liver-specific function by mouse fibroblast nuclei transplanted into rat hepatoma cytoplasts, Nature, 281: 74.PubMedCrossRefGoogle Scholar
  22. Martin, G.M., 1977, Cellular aging - clonal senescence, Amer. J. Path., 89: 484.Google Scholar
  23. Mitcheson, J.M., 1971, The Biology of the Cell Cycle, Cambridge University Press, London, pp. 29–33, 244–249.Google Scholar
  24. Mitsui, Y. and Schneider, E.L., 1976, Increased nuclear sizes in senescent human diploid fibroblast cultures, Exp. Cell. Res., 100: 147.Google Scholar
  25. Muggleton-Harris, A.L. and Hayflick, L., 1976, Cellular aging studied by the reconstruction’of replicating cells from nuclei and cytoplasms isolated from normal human diploid cells, Exp. Cell. Res., 103: 321.Google Scholar
  26. Muggleton-Harris, A.L. and DeSimone, D.W., 1980, Replicative potentials of various fusion products between WI-38 and SV-40 transformed WI-38 cells and their components, Somat. Cell Genet. 6: 689.Google Scholar
  27. Muggleton-Harris, A.L. and Aroian, M.A., 1982, Replicative potential of individual cell hybrids derived from young and old donor human skin fibroblasts, Somat. Cell Genet. 8: 41.Google Scholar
  28. Nette, E.G., Sit, H.L., King, D.W., 1982, Reactivation of DNA synthesis in aging diploid human skin fibroblasts by fusion with mouse L karyoplasts, cytoplasts and whole L cells, Mech. Age. Dev. 18: 75.Google Scholar
  29. Noguchi, H., Reddy, G.P., and Pardee, A.B., 1983, Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei, Cell 32: 443.PubMedCrossRefGoogle Scholar
  30. Norwood, T.H., Hoehn, H., Salk, D., and Martin, G.M., 1979a, Cellular aging in Werner’s syndrome: A unique phenotype, J. Invest. Dermatol. 72: 92.Google Scholar
  31. Norwood, T.H., Pendergrass, W.R., and Martin, G.M., 1975, Reinitiation of DNA synthesis in senescent human fibroblasts upon fusion with cells of unlimited growth potential, J. Cell Biol. 64: 551.PubMedCrossRefGoogle Scholar
  32. Norwood, T.H., Pendergrass, W., Bornstein, P., and Martin, G.M., 1979b, DNA synthesis of sublethally injured cells in heterokaryons and its relevance to clonal senescence, Exp. Cell Res. 119: 15.Google Scholar
  33. Norwood, T.H., Pendergrass, W.R., Sprague, C.A., and Martin, G.M., 1974, Dominance of the senescent phenotype in heterokaryons between replicative and post-replicative human fibroblast-like cells, Proc. Natl. Acad. Sci., 73: 223.Google Scholar
  34. Norwood, T.H. and Smith, J.R., 1985, “The cultural fibroblast-like cell as a model for the study of aging.” In Handbook of the Biology of Aging, Eds., C.E. Finch and E.L. Schneider, Van Nostrond Reinhold, New York, in press.Google Scholar
  35. Norwood, T.H., Zeigler, C.J., 1979c, Complementation between senescent human diploid cells and a thymidine kinase deficient murine cell line, Cytogenet. Cell Genet. 19: 355.Google Scholar
  36. Orgel, L.E., 1963, The maintenance of the accuracy of protein synthesis and its relevance to aging, Proc. Natl. Acad. Sci., 49: 517.Google Scholar
  37. Orgel, L.E., 1970, The maintenance of the accuracy of protein synthesis and its relevance to aging: A correction, Proc. Natl. Acad. Sci., 67: 1476.Google Scholar
  38. Pendergrass, W.R., Saulewicz, A.C., Burmer, G.C., Rabinovitch, P.S., Norwood, T.H., and Martin, G.M., 1982, Evidence that a critical threshold of DNA synthesis in mammalian cells heterokaryons, J. Cell. Physiol. 133: 141.Google Scholar
  39. Pereira-Smith, O.M. and Smith, J.R., 1981, Expression of SV-40 T antigen in finite life-span hybrids of normal and SV-40¬transformed fibroblasts. Somat. Cell. Genet. 7: 411.Google Scholar
  40. Pereira-Smith, O.M. and Smith, J.R., 1982, The phenotype of low proliferative potential is dominant in hybrids of normal human fibroblasts. Somat. Cell Genet..Google Scholar
  41. Rabinovitch, P.S. and Norwood, T.H., 1980, Comparative heterokaryon study of cellular senescence and the serum-deprived state. Exp. Cell. Res. 130: 101.Google Scholar
  42. Rao, M.V.N., 1976, Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells, Exp. Cell Res., 102: 25.Google Scholar
  43. Rao, P.N., Satya-Prakash,K.L. 1983, Inducers of DNA synthesis: Levels higher in transformed cells than in normal cells, J. Cell Biol. 96: 571.Google Scholar
  44. Reddy, P.V. and Pardee, A.G., 1980, Multienzyme complex for metabolic channeling in mammalian DNA replication, Proc. Natl. Acad. Sci., USA, 77: 3312.Google Scholar
  45. Salk, D., 1982, Werner’s syndrome: A review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells and chromosomal aberrations, Human Genetics, 62: 1.Google Scholar
  46. Salk, D., Bryant, E., Au, K., Hoehn, H., and Martin, G.M., 1981, Systematic growth studies, cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts, Hum. Genet. 58: 310.Google Scholar
  47. Scappaticci, S., Cerimele, D., Fraccaro, M., 1982, Clonal structural chromosomal rearrangements in primary fibroblast cultures and in lymphocytes of patients with Werner’s syndrome, Hum. Genet. 62: 16.Google Scholar
  48. Schonberg, S.A., Henderson, E., Niermeijer, M.F., and German, J., 1981, Werner’s syndrome: preferential proliferation of clones with translocations, Am. J. Hum. Genet. 33: 120A.Google Scholar
  49. Stanbridge, E.J., 1976, Suppression of malignancy in human cells, Nature, 260: 17.PubMedCrossRefGoogle Scholar
  50. Stein, G.H. and Yanishevsky, R.M., 1981, Quiescent human diploid cells can inhibit entry into S phase in replicative nuclei in heterodikaryons, Proc. Natl. Acad. Sci. 78: 325.Google Scholar
  51. Stein, G.H. and Yanishevsky, R.M., 1979, Entry into S phase is inhibited in two immortal cell lines fused to senescent human diploid cells, Exp. Cell. Res. 120: 155.Google Scholar
  52. Stein, G.H., Yanishevsky, R.M., Gordon, L., and Beeson, M., 1982, 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. 79: 5287.Google Scholar
  53. Tanaka, K., Nakazawa, T., Okada, Y., and Kmahara, Y., 1979, Increase in DNA synthesis in Werner’s syndrome cells by hybridization with normal human diploid and hela cells, Exp. Cell Res. 123:261.Google Scholar
  54. Tanaka, K., Nakazawa, T., Okada, Y., and Kumahara, Y., 1980, Roles of nuclear and cytoplasmic environments in the retarded DNA synthesis in Werner’s syndrome cells, Exp. Cell Res. 127: 185.Google Scholar
  55. Wright, W.E. and Hayflick, L., 1975a, Nuclear control of cellular aging demonstrated by hybridization of anucleate and whole cultured normal human fibroblasts, Exp. Cell Res. 96: 113.Google Scholar
  56. Wright, W.E. and Hayflick, L., 1975b, The regulation of cellular aging by nuclear events in cultured normal fibroblasts (WI-38), Adv. Exp. Mol. Biol, 61: 39.Google Scholar
  57. Yanishevsky, R.M. and Stein, G.H., 1981, Regulation of the cell cycle in eukaryotyic cells, Int. Rev. Cytol. 69: 223.Google Scholar
  58. Yanishevsky, R.M. and Stein, G.H., 1980, 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: 469Google Scholar

Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • William Pendergrass
    • 1
  • Darrell Salk
    • 1
  • Thomas Norwood
    • 1
  1. 1.Department of PathologyUniversity of WashingtonSeattleUSA

Personalised recommendations