Cytotechnology

, Volume 45, Issue 1–2, pp 33–38 | Cite as

Use of exogenous hTERT to immortalize primary human cells

  • Kwang M. Lee
  • Kyung H. Choi
  • Michel M. Ouellette
Article

Abstract

A major obstacle to the immortalization of primary human cells and the establishment of human cell lines is telomere-controlled senescence. Telomere-controlled senescence is caused by the shortening of telomeres that occurs each time somatic human cells divide. The enzyme telomerase can prevent the erosion of telomeres and block the onset of telomere-controlled senescence, but its expression is restricted to the early stages of embryonic development, and in the adult, to rare cells of the blood, skin and digestive track. However, we and others have shown that the transfer of an exogenous hTERT cDNA, encoding the catalytic subunit of human telomerase, can be used to prevent telomere shortening, overcome telomere-controlled senescence, and immortalize primary human cells. Most importantly, hTERT alone can immortalize cells without causing cancer-associated changes or altering phenotypic properties. Primary human cells that have so far been established by the forced expression of hTERT alone include fibroblasts, retinal pigmented epithelial cells, endothelial cells, oesophageal squamous cells, mammary epithelial cells, keratinocytes, osteoblasts, and Nestin-positive cells of the pancreas. In this article, we discuss the use of hTERT to immortalize of human cells, the properties of hTERT-immortalized cells, and their applications to cancer research and tissue engineering.

Keywords

Cancer-associated changes Checkpoint hTERT hTR Immortalization Oncogene Senescence Telomerase Telomere 

Abbreviations

hTERT

human Telomerase Reverse Transcriptase

hTR -

human Telomerase RNA.

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References

  1. Allsopp, R.C., Harley, C.B. 1995Evidence for a critical telomere length in senescent human fibroblastsExp. Cell Res.219130136CrossRefPubMedGoogle Scholar
  2. Blackburn, E.H. 1994Telomeres: no end in sightCell77621623CrossRefGoogle Scholar
  3. Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., Wright, W.E. 1998Extension of life-span by introduction of telomerase into normal human cellsScience279349352PubMedGoogle Scholar
  4. Brenner, A.J., Stampfer, M.R., Aldaz, C.M. 1998Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivationOncogene17199205CrossRefPubMedGoogle Scholar
  5. Condon, J., Yin, S., Mayhew, B., Word, R.A., Wright, W.E., Shay, J.W., Rainey, W.E. 2002Telomerase immortalization of human myometrial cells Biol. Reprod.67506514PubMedGoogle Scholar
  6. Counter, C.M., Avilion, A.A., LeFeuvre, C.E., Stewart, N.G., Greider, C.W., Harley, C.B., Bacchetti, S. 1992Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activityEmbo. J.1119211929PubMedGoogle Scholar
  7. Counter, C.M., Botelho, F.M., Wang, P., Harley, C.B., Bacchetti, S. 1994Stabilization of short telomeres and telomerase activity accompany immortalization of epstein-barr virus-transformed human b lymphocytesJ. Virol.6834103414PubMedGoogle Scholar
  8. Counter, C.M., Hahn, W.C., Wei, W., Caddle, S.D., Beijersbergen, R.L., Lansdorp, P.M., Sedivy, J.M., Weinberg, R.A. 1998Dissociation among in vitro telomerase activity, telomere maintenanceand cellular immortalizationProc. Natl. Acad. Sci. USA951472314728CrossRefPubMedGoogle Scholar
  9. de Lange, T. 2002Protection of mammalian telomeresOncogene21532540CrossRefPubMedGoogle Scholar
  10. Di Leonardo, A., Linke, S.P., Clarkin, K., Wahl, G.M. 1994DNA damage triggers a prolonged p53-dependent g1 arrest and long-term induction of cip1 in normal human fibroblastsGene. Dev.825402551PubMedGoogle Scholar
  11. Farwell, D.G., Shera, K.A., Koop, J.I., Bonnet, G.A., Matthews, C.P., Reuther, G.W., Coltrera, M.D., McDougall, J.K., Klingelhutz, A.J. 2000Genetic and epigenetic changes in human epithelial cells immortalized by telomeraseAm. J. Pathol.15615371547PubMedGoogle Scholar
  12. Feng, J., Funk, W.D., Wang, S.S., Weinrich, S.L., Avilion, A.A., Chiu, C.P., Adams, R.R., Chang, E., Allsopp, R.C., Yu, J. 1995The RNA component of human telomeraseScience26912361241PubMedGoogle Scholar
  13. Harrington, L., Zhou, W., McPhail, T., Oulton, R., Yeung, D.S., Mar, V., Bass, M.B., Robinson, M.O.,  et al. 1997Human telomerase contains evolutionarily conserved catalytic and structural subunitsGene. Dev.1131093115PubMedGoogle Scholar
  14. Hayflick, L., Moorhead, P.S. 1961The serial cultivation of human diploid cell strainsExp. Cell Res.25585621CrossRefGoogle Scholar
  15. Herbert, B.S., Wright, W.E., Shay, J.W. 2002P16(ink4a) inactivation is not required to immortalize human mammary epithelial cellsOncogene2178977900CrossRefPubMedGoogle Scholar
  16. Jiang, X.R., Jimenez, G., Chang, E., Frolkis, M., Kusler, B., Sage, M., Beeche, M., Bodnar, A.G., Wahl, G.M., Tlsty, T.D., Chiu, C.P. 1999Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotypeNat. Genet.21111114CrossRefPubMedGoogle Scholar
  17. Kilian, A., Bowtell, D.D., Abud, H.E., Hime, G.R., Venter, D.J., Keese, P.K., Duncan, E.L., Reddel, R.R., Jefferson, R.A. 1997Isolation of a candidate human telomerase catalytic subunit genewhich reveals complex splicing patterns in different cell typesHum. Mol. Genet.620112019CrossRefPubMedGoogle Scholar
  18. Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L., Coviello, G.M., Wright, W.E., Weinrich, S.L., Shay, J.W. 1994Specific association of human telomerase activity with immortal cells and cancerScience26620112015PubMedGoogle Scholar
  19. Kiyono, T., Foster, S.A., Koop, J.I., McDougall, J.K., Galloway, D.A., Klingelhutz, A.J. 1998Both pRB/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells Nature3968488CrossRefPubMedGoogle Scholar
  20. Lee, K.M., Nguyen, C., Ulrich, A.B., Pour, P.M., Ouellette, M.M. 2003Immortalization with telomerase of the nestin-positive cells of the human pancreasBiochem. Biophys. Res. Commun.30110381044CrossRefPubMedGoogle Scholar
  21. Masutomi, K., Yu, E.Y., Khurts, S., Ben-Porath, I., Currier, J.L., Metz, G.B., Brooks, M.W., Kaneko, S., Murakami, S., DeCaprio, J.A., Weinberg, R.A., Stewart, S.A., Hahn, W.C. 2003Telomerase maintains telomere structure in normal human cellsCell114241253CrossRefPubMedGoogle Scholar
  22. Meyerson, M., Counter, C.M., Eaton, E.N., Ellisen, L.W., Steiner, P., Caddle, S.D., Ziaugra, L., Beijersbergen, R.L., Davidoff, M.J., Liu, Q., Bacchetti, S., Haber, D.A., Weinberg, R.A. 1997Hest2, the putative human telomerase catalytic subunit geneis up-regulated in tumor cells and during immortalizationCell90785795PubMedGoogle Scholar
  23. Morales, C.P., Holt, S.E., Ouellette, M., Kaur, K.J., Yan, Y., Wilson, K.S., White, M.A., Wright, W.E., Shay, J.W. 1999Absence of cancer-associated changes in human fibroblasts immortalized with telomeraseNat. Genet.21115118CrossRefPubMedGoogle Scholar
  24. Morales, C.P., Gandia, K.G., Ramirez, R.D., Wright, W.E., Shay, J.W., Spechler, S.J. 2003Characterisation of telomerase immortalised normal human oesophageal squamous cellsGut52327333CrossRefPubMedGoogle Scholar
  25. Nakamura, T.M., Morin, G.B., Chapman, K.B., Weinrich, S.L., Andrews, W.H., Lingner, J., Harley, C.B., Cech, T.R. 1997Telomerase catalytic subunit homologs from fission yeast and humanScience277955959Google Scholar
  26. Ouellette, M.M., McDaniel, L.D., Wright, W.E., Shay, J.W., Schultz, R.A. 2000The establishment of telomerase-immortalized cell lines representing human chromosome instability syndromesHum. Mol. Genet.9403411CrossRefPubMedGoogle Scholar
  27. Ouellette, M.M., Lee, K. 2001Telomerase: diagnostics, cancer therapeutics and tissue engineeringDrug Discov. Today612311237CrossRefPubMedGoogle Scholar
  28. Ramirez, R.D., Morales, C.P., Herbert, B.S., Rohde, J.M., Passons, C., Shay, J.W., Wright, W.E. 2001Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditionsGene. Dev.15398403CrossRefPubMedGoogle Scholar
  29. Robles, S.J., Adami, G.R. 1998Agents that cause DNA double strand breaks lead to p16ink4a enrichment and the premature senescence of normal fibroblastsOncogene1611131123CrossRefPubMedGoogle Scholar
  30. Romanov, S.R., Kozakiewicz, B.K., Holst, C.R., Stampfer, M.R., Haupt, L.M., Tlsty, T.D. 2001Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changesNature409633637CrossRefPubMedGoogle Scholar
  31. Shay, J.W., Pereira-Smith, O.M., Wright, W.E. 1991A role for both rb and p53 in the regulation of human cellular senescenceExp. Cell Res.1963339CrossRefPubMedGoogle Scholar
  32. Shay, J.W., West, M.D., Wright, W.E. 1992Re-expression of senescent markers in deinduced reversibly immortalized cellsExp. Gerontol.27477492CrossRefPubMedGoogle Scholar
  33. Shay, J.W., Der Haegen, B.A., Ying, Y., Wright, W.E. 1993The frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with sv40 large t-antigenExp. Cell Res.2094552CrossRefPubMedGoogle Scholar
  34. Shay, J.W., Wright, W.E. 1996Telomerase activity in human cancerCurr. Opin. Oncol.86671PubMedGoogle Scholar
  35. Shay, J.W., Bacchetti, S. 1997A survey of telomerase activity in human cancerEur. J. Cancer33787791CrossRefPubMedGoogle Scholar
  36. Shelton, D.N., Chang, E., Whittier, P.S., Choi, D., Funk, W.D. 1999Microarray analysis of replicative senescenceCurr. Biol.9939945CrossRefPubMedGoogle Scholar
  37. Smogorzewska, A., de Lange, T. 2002Different telomere damage signaling pathways in human and mouse cellsEmbo. J.2143384348CrossRefPubMedGoogle Scholar
  38. Vaziri, H., Benchimol, S. 1998Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life spanCurr. Biol.8279282CrossRefPubMedGoogle Scholar
  39. Wright, W.E., Shay, J.W. 1992The two-stage mechanism controlling cellular senescence and immortalizationExp. Gerontol.27383389CrossRefPubMedGoogle Scholar
  40. Yang, J., Chang, E., Cherry, A.M., Bangs, C.D., Oei, Y., Bodnar, A., Bronstein, A., Chiu, C.P., Herron, G.S. 1999Human endothelial cell life extension by telomerase expressionJ. Biol. Chem.2742614126148CrossRefPubMedGoogle Scholar
  41. Yudoh, K., Matsuno, H., Nakazawa, F., Katayama, R., Kimura, T. 2001Reconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblastsJ. Bone Miner. Res.1614531464PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Kwang M. Lee
    • 1
  • Kyung H. Choi
    • 1
  • Michel M. Ouellette
    • 1
  1. 1.Eppley Institute for Research in Cancer and Allied DiseasesUniversity of Nebraska Medical CenterOmahaUSA

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