Biogerontology

, Volume 8, Issue 2, pp 163–172 | Cite as

Telomerase activity in HeLa cervical carcinoma cell line proliferation

  • Milena Ivanković
  • Andrea Ćukušić
  • Ivana Gotić
  • Nikolina Škrobot
  • Mario Matijašić
  • Denis Polančec
  • Ivica Rubelj
Research Article

Abstract

Normal human somatic cells in culture have a limited dividing potential. This is due to DNA end replication problem, whereby telomeres shorten with each subsequent cell division. When a critical telomere length is reached cells enter senescence. To overcome this problem, immortal HeLa cell line express telomerase, an enzyme that prevents telomere shortening. Although immortal, the existence of non-dividing cells that do not incorporate 3H-thymidine over 24 h of growth has been well documented in this cell line. Using DiI labeling and high-speed cell sorting, we have separated and analyzed fractions of HeLa cells that divided vigorously as well as those that cease divisions over several days in culture. We also analyzed telomerase activity in separated fractions and surprisingly, found that the fraction of cells that divided 0–1 time over 6 days in culture have several times higher endogenous telomerase activity than the fastest dividing fraction. Additionally, the non-growing fraction regains an overall high labeling index and low SA-β-Gal activity when subcultured again. This phenomenon should be considered if telomerase inhibition is to be used as an approach to cancer therapy. In this paper we also discuss possible molecular mechanisms that underlie the observed results.

Keywords

DiI Flow cytometry HeLa Senescence Telomerase 

Notes

Acknowledgments

We thank Dr. Olivia Pereira-Smith, Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio for reviewing the manuscript. This work was supported by Croatian Ministry of Science, Education and Sports grant 0098077.

References

  1. Bryan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR (1995) Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 14:4240–4248PubMedGoogle Scholar
  2. Bryan TM, Englezou A, Dunham M, Reddel RR (1998) Telomere length dynamics in telomerase-positive immortal human cell populations. Exp Cell Res 239:370–378PubMedCrossRefGoogle Scholar
  3. Campisi J (1992) Gene expression in quiescent and senescent fibroblasts. Ann NY Acad Sci 21:195–201CrossRefGoogle Scholar
  4. Campisi J (2000) Cancer, aging and cellular senescence. In Vivo 14:183–188PubMedGoogle Scholar
  5. Corey RD (2002) Telomerase inhibition, oligonucleotides, and clinical trials. Oncogene 21:631–637PubMedCrossRefGoogle Scholar
  6. Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S (1992) Telomere shortening associated with chromosome instability is arrested in immortal cell which express telomerase activity. EMBO J 11:1921–1929PubMedGoogle Scholar
  7. Cristofalo VJ, Pignolo RJ, Rotenberg MO (1992) Molecular changes with in vitro cellular senescence. Ann NY Acad Sci 663:187–194PubMedCrossRefGoogle Scholar
  8. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith OM (1995) A biomarker that identifies senescent human cells in culture and aging skin in vivo. Proc Natl Acad Sci USA 92:9363–9367PubMedCrossRefGoogle Scholar
  9. Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, Adams RR, Chang E, Allsopp RC, Yu J, Le S, West MD, Harley CB, Andrews WH, Greider CW, Villeponteau B (1995) The RNA component of human telomerase. Science 269:1236–1241PubMedCrossRefGoogle Scholar
  10. Ferenac M, Polancec D, Huzak M, Pereira-Smith OM, Rubelj I (2005) Early-senescing human skin fibroblasts do not demonstrate accelerated telomere shortening. J Gerontol A Biol Sci Med Sci 60:820–829PubMedGoogle Scholar
  11. Goodwin EC, DiMaio D (2001) Induced senescence in HeLa cervical carcinoma cells containing elevated telomerase activity and extended telomeres. Cell Growth Differ 11:525–534Google Scholar
  12. Gorbunova V, Seluanov A, Pereira-Smith OM (2003) Evidence that high telomerase activity may induce a senescent-like growth arrest in human fibroblasts. J Biol Chem 287:7692–7698CrossRefGoogle Scholar
  13. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblast. Nature 345:458–460PubMedCrossRefGoogle Scholar
  14. Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 37:614–636PubMedCrossRefGoogle Scholar
  15. Hayflick L, Moorhead PS (1961) The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 25:585–621CrossRefGoogle Scholar
  16. Hemann MT, Strong MA, Hao LY, Greider CW (2001) The shortest telomere, not average telomere length is critical for cell viability and chromosome stability. Cell 107:67–77PubMedCrossRefGoogle Scholar
  17. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW (1994) Specific association of human telomerase activity in immortal cells and cancer. Science 266:2011–2015PubMedCrossRefGoogle Scholar
  18. Ledley FD, Soriano HE, O’Malley BW Jr, Lewis D, Darlington GJ, Finegold M (1992) DiI as a marker for cellular transplantation into solid organs. Biotechniques 13:580–587PubMedGoogle Scholar
  19. Makarov VL, Hirose Y, Langmore JP (1997) Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 88:657–666PubMedCrossRefGoogle Scholar
  20. Martinez AO, Norwood TH, Prothero JW, Martin GM (1978) Evidence for clonal attenuation of growth potential in HeLa cells. In Vitro 14:996–1002PubMedGoogle Scholar
  21. Matsumura T, Pfendt EA, Hayflick L (1979) DNA synthesis in the human diploid cell strain WI-38 during in vitro aging: an autoradiography study. J Gerontol 34:323–327PubMedGoogle Scholar
  22. Morin GB (1989) The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59:521–529PubMedCrossRefGoogle Scholar
  23. Neumann AA, Reddel RR (2002) Telomere maintenance and cancer—look, no telomerase. Nat Rev Cancer 2:879–884PubMedCrossRefGoogle Scholar
  24. Olovnikov AM (1973) The incomplete copying of template margin in enzymatic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 41:181–190PubMedCrossRefGoogle Scholar
  25. Pereira-Smith OM, Smith JR (1981) Expression of SV40 T antigen in finite life-span hybrids of normal and SV40-transformed fibroblasts. Somatic Cell Genet 7:411–421PubMedCrossRefGoogle Scholar
  26. Roninson IB (2003) Tumor cell senescence in cancer treatment. Can Res 63:2705–2715Google Scholar
  27. Rubelj I, Venable SF, Lednicky J, Butel JS, Bilyeu T, Darlington G, Surmacz E, Campisi J, Pereira-Smith OM (1997) Loss of T-antigen sequences allows SV40-transformed human cells in crisis to acquire a senescent-like phenotype. J Gerontol A Biol Sci Med Sci 52:229–234Google Scholar
  28. Rubelj I, Huzak M, Brdar B, Pereira-Smith OM (2002) A single stage mechanism controls replicative senescence through Sudden Senescence Syndrome. Biogerontology 3:213–222PubMedCrossRefGoogle Scholar
  29. Saretzki G, Von Zglinicki T (2002) Replicative aging, telomeres and oxidative stress. Ann NY Acad Sci 959:24–29PubMedCrossRefGoogle Scholar
  30. Shay JW, Bacchetti S (1997) A survey of telomerase activity in human cancer. Eur J Cancer 33:787–791PubMedCrossRefGoogle Scholar
  31. Te Poele RH, Ohorkov AL, Jardine L, Cummings J, Joel SP (2002) DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res 62:1876–1883PubMedGoogle Scholar
  32. Touissaint O, Medrano EE, Von Zglinicki T (2000) Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. Exp Gerontol 35:927–945CrossRefGoogle Scholar
  33. Von Zglinicki T, Nilsson E, Docke WD, Brunk UT (1995) Lipofuscin accumulation and ageing of fibroblasts. Gerontology 41:95–108CrossRefGoogle Scholar
  34. Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW (1996) Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 18:173–179PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Milena Ivanković
    • 1
  • Andrea Ćukušić
    • 1
  • Ivana Gotić
    • 1
  • Nikolina Škrobot
    • 1
  • Mario Matijašić
    • 2
  • Denis Polančec
    • 2
  • Ivica Rubelj
    • 1
  1. 1.Department of Molecular BiologyRuđer Bošković InstituteZagrebCroatia
  2. 2.Biology, Flow Cytometry and Cell Sorting LabPLIVA—Research Institute LtdZagrebCroatia

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