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Use of exogenous hTERT to immortalize primary human cells

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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.

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Abbreviations

hTERT:

human Telomerase Reverse Transcriptase

hTR -:

human Telomerase RNA.

References

  • R.C. Allsopp C.B. Harley (1995) ArticleTitleEvidence for a critical telomere length in senescent human fibroblasts Exp. Cell Res. 219 130–136 Occurrence Handle10.1006/excr.1995.1213 Occurrence Handle7628529

    Article  PubMed  Google Scholar 

  • E.H. Blackburn (1994) ArticleTitleTelomeres: no end in sight Cell 77 621–623 Occurrence Handle10.1016/0092-8674(94)90046-9

    Article  Google Scholar 

  • A.G. Bodnar M. Ouellette M. Frolkis S.E. Holt C.P. Chiu G.B. Morin C.B. Harley J.W. Shay S. Lichtsteiner W.E. Wright (1998) ArticleTitleExtension of life-span by introduction of telomerase into normal human cells Science 279 349–352 Occurrence Handle9454332

    PubMed  Google Scholar 

  • A.J. Brenner M.R. Stampfer C.M. Aldaz (1998) ArticleTitleIncreased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation Oncogene 17 199–205 Occurrence Handle10.1038/sj.onc.1201919 Occurrence Handle9674704

    Article  PubMed  Google Scholar 

  • J. Condon S. Yin B. Mayhew R.A. Word W.E. Wright J.W. Shay W.E. Rainey (2002) ArticleTitleTelomerase immortalization of human myometrial cells Biol. Reprod. 67 506–514 Occurrence Handle12135889

    PubMed  Google Scholar 

  • C.M. Counter A.A. Avilion C.E. LeFeuvre N.G. Stewart C.W. Greider C.B. Harley S. Bacchetti (1992) ArticleTitleTelomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity Embo. J. 11 1921–1929 Occurrence Handle1582420

    PubMed  Google Scholar 

  • C.M. Counter F.M. Botelho P. Wang C.B. Harley S. Bacchetti (1994) ArticleTitleStabilization of short telomeres and telomerase activity accompany immortalization of epstein-barr virus-transformed human b lymphocytes J. Virol. 68 3410–3414 Occurrence Handle8151802

    PubMed  Google Scholar 

  • C.M. Counter W.C. Hahn W. Wei S.D. Caddle R.L. Beijersbergen P.M. Lansdorp J.M. Sedivy R.A. Weinberg (1998) ArticleTitleDissociation among in vitro telomerase activity, telomere maintenanceand cellular immortalization Proc. Natl. Acad. Sci. USA 95 14723–14728 Occurrence Handle10.1073/pnas.95.25.14723 Occurrence Handle9843956

    Article  PubMed  Google Scholar 

  • T. de Lange (2002) ArticleTitleProtection of mammalian telomeres Oncogene 21 532–540 Occurrence Handle10.1038/sj.onc.1205080 Occurrence Handle11850778

    Article  PubMed  Google Scholar 

  • A. Di Leonardo S.P. Linke K. Clarkin G.M. Wahl (1994) ArticleTitleDNA damage triggers a prolonged p53-dependent g1 arrest and long-term induction of cip1 in normal human fibroblasts Gene. Dev. 8 2540–2551 Occurrence Handle7958916

    PubMed  Google Scholar 

  • D.G. Farwell K.A. Shera J.I. Koop G.A. Bonnet C.P. Matthews G.W. Reuther M.D. Coltrera J.K. McDougall A.J. Klingelhutz (2000) ArticleTitleGenetic and epigenetic changes in human epithelial cells immortalized by telomerase Am. J. Pathol. 156 1537–1547 Occurrence Handle10793065

    PubMed  Google Scholar 

  • J. Feng W.D. Funk S.S. Wang S.L. Weinrich A.A. Avilion C.P. Chiu R.R. Adams E. Chang R.C. Allsopp J. Yu (1995) ArticleTitleThe RNA component of human telomerase Science 269 1236–1241 Occurrence Handle7544491

    PubMed  Google Scholar 

  • L. Harrington W. Zhou T. McPhail R. Oulton D.S. Yeung V. Mar M.B. Bass M.O. Robinson et al. (1997) ArticleTitleHuman telomerase contains evolutionarily conserved catalytic and structural subunits Gene. Dev. 11 3109–3115 Occurrence Handle9389643

    PubMed  Google Scholar 

  • L. Hayflick P.S. Moorhead (1961) ArticleTitleThe serial cultivation of human diploid cell strains Exp. Cell Res. 25 585–621 Occurrence Handle10.1016/0014-4827(61)90192-6

    Article  Google Scholar 

  • B.S. Herbert W.E. Wright J.W. Shay (2002) ArticleTitleP16(ink4a) inactivation is not required to immortalize human mammary epithelial cells Oncogene 21 7897–7900 Occurrence Handle10.1038/sj.onc.1205902 Occurrence Handle12420227

    Article  PubMed  Google Scholar 

  • X.R. Jiang G. Jimenez E. Chang M. Frolkis B. Kusler M. Sage M. Beeche A.G. Bodnar G.M. Wahl T.D. Tlsty C.P. Chiu (1999) ArticleTitleTelomerase expression in human somatic cells does not induce changes associated with a transformed phenotype Nat. Genet. 21 111–114 Occurrence Handle10.1038/5056 Occurrence Handle9916802

    Article  PubMed  Google Scholar 

  • A. Kilian D.D. Bowtell H.E. Abud G.R. Hime D.J. Venter P.K. Keese E.L. Duncan R.R. Reddel R.A. Jefferson (1997) ArticleTitleIsolation of a candidate human telomerase catalytic subunit genewhich reveals complex splicing patterns in different cell types Hum. Mol. Genet. 6 2011–2019 Occurrence Handle10.1093/hmg/6.12.2011 Occurrence Handle9328464

    Article  PubMed  Google Scholar 

  • N.W. Kim M.A. Piatyszek K.R. Prowse C.B. Harley M.D. West P.L. Ho G.M. Coviello W.E. Wright S.L. Weinrich J.W. Shay (1994) ArticleTitleSpecific association of human telomerase activity with immortal cells and cancer Science 266 2011–2015 Occurrence Handle7605428

    PubMed  Google Scholar 

  • T. Kiyono S.A. Foster J.I. Koop J.K. McDougall D.A. Galloway A.J. Klingelhutz (1998) ArticleTitleBoth pRB/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells Nature 396 84–88 Occurrence Handle10.1038/23962 Occurrence Handle9817205

    Article  PubMed  Google Scholar 

  • K.M. Lee C. Nguyen A.B. Ulrich P.M. Pour M.M. Ouellette (2003) ArticleTitleImmortalization with telomerase of the nestin-positive cells of the human pancreas Biochem. Biophys. Res. Commun. 301 1038–1044 Occurrence Handle10.1016/S0006-291X(03)00086-X Occurrence Handle12589817

    Article  PubMed  Google Scholar 

  • K. Masutomi E.Y. Yu S. Khurts I. Ben-Porath J.L. Currier G.B. Metz M.W. Brooks S. Kaneko S. Murakami J.A. DeCaprio R.A. Weinberg S.A. Stewart W.C. Hahn (2003) ArticleTitleTelomerase maintains telomere structure in normal human cells Cell 114 241–253 Occurrence Handle10.1016/S0092-8674(03)00550-6 Occurrence Handle12887925

    Article  PubMed  Google Scholar 

  • M. Meyerson C.M. Counter E.N. Eaton L.W. Ellisen P. Steiner S.D. Caddle L. Ziaugra R.L. Beijersbergen M.J. Davidoff Q. Liu S. Bacchetti D.A. Haber R.A. Weinberg (1997) ArticleTitleHest2, the putative human telomerase catalytic subunit geneis up-regulated in tumor cells and during immortalization Cell 90 785–795 Occurrence Handle9288757

    PubMed  Google Scholar 

  • C.P. Morales S.E. Holt M. Ouellette K.J. Kaur Y. Yan K.S. Wilson M.A. White W.E. Wright J.W. Shay (1999) ArticleTitleAbsence of cancer-associated changes in human fibroblasts immortalized with telomerase Nat. Genet. 21 115–118 Occurrence Handle10.1038/5063 Occurrence Handle9916803

    Article  PubMed  Google Scholar 

  • C.P. Morales K.G. Gandia R.D. Ramirez W.E. Wright J.W. Shay S.J. Spechler (2003) ArticleTitleCharacterisation of telomerase immortalised normal human oesophageal squamous cells Gut 52 327–333 Occurrence Handle10.1136/gut.52.3.327 Occurrence Handle12584211

    Article  PubMed  Google Scholar 

  • T.M. Nakamura G.B. Morin K.B. Chapman S.L. Weinrich W.H. Andrews J. Lingner C.B. Harley T.R. Cech (1997) ArticleTitleTelomerase catalytic subunit homologs from fission yeast and human Science 277 955–959

    Google Scholar 

  • M.M. Ouellette L.D. McDaniel W.E. Wright J.W. Shay R.A. Schultz (2000) ArticleTitleThe establishment of telomerase-immortalized cell lines representing human chromosome instability syndromes Hum. Mol. Genet. 9 403–411 Occurrence Handle10.1093/hmg/9.3.403 Occurrence Handle10655550

    Article  PubMed  Google Scholar 

  • M.M. Ouellette K. Lee (2001) ArticleTitleTelomerase: diagnostics, cancer therapeutics and tissue engineering Drug Discov. Today 6 1231–1237 Occurrence Handle10.1016/S1359-6446(01)02052-9 Occurrence Handle11722875

    Article  PubMed  Google Scholar 

  • R.D. Ramirez C.P. Morales B.S. Herbert J.M. Rohde C. Passons J.W. Shay W.E. Wright (2001) ArticleTitlePutative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions Gene. Dev. 15 398–403 Occurrence Handle10.1101/gad.859201 Occurrence Handle11230148

    Article  PubMed  Google Scholar 

  • S.J. Robles G.R. Adami (1998) ArticleTitleAgents that cause DNA double strand breaks lead to p16ink4a enrichment and the premature senescence of normal fibroblasts Oncogene 16 1113–1123 Occurrence Handle10.1038/sj.onc.1201862 Occurrence Handle9528853

    Article  PubMed  Google Scholar 

  • S.R. Romanov B.K. Kozakiewicz C.R. Holst M.R. Stampfer L.M. Haupt T.D. Tlsty (2001) ArticleTitleNormal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes Nature 409 633–637 Occurrence Handle10.1038/35054579 Occurrence Handle11214324

    Article  PubMed  Google Scholar 

  • J.W. Shay O.M. Pereira-Smith W.E. Wright (1991) ArticleTitleA role for both rb and p53 in the regulation of human cellular senescence Exp. Cell Res. 196 33–39 Occurrence Handle10.1016/0014-4827(91)90453-2 Occurrence Handle1652450

    Article  PubMed  Google Scholar 

  • J.W. Shay M.D. West W.E. Wright (1992) ArticleTitleRe-expression of senescent markers in deinduced reversibly immortalized cells Exp. Gerontol. 27 477–492 Occurrence Handle10.1016/0531-5565(92)90003-I Occurrence Handle1330670

    Article  PubMed  Google Scholar 

  • J.W. Shay B.A. Der Haegen Y. Ying W.E. Wright (1993) ArticleTitleThe frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with sv40 large t-antigen Exp. Cell Res. 209 45–52 Occurrence Handle10.1006/excr.1993.1283 Occurrence Handle8224005

    Article  PubMed  Google Scholar 

  • J.W. Shay W.E. Wright (1996) ArticleTitleTelomerase activity in human cancer Curr. Opin. Oncol. 8 66–71 Occurrence Handle8868103

    PubMed  Google Scholar 

  • J.W. Shay S. Bacchetti (1997) ArticleTitleA survey of telomerase activity in human cancer Eur. J. Cancer 33 787–791 Occurrence Handle10.1016/S0959-8049(97)00062-2 Occurrence Handle9282118

    Article  PubMed  Google Scholar 

  • D.N. Shelton E. Chang P.S. Whittier D. Choi W.D. Funk (1999) ArticleTitleMicroarray analysis of replicative senescence Curr. Biol. 9 939–945 Occurrence Handle10.1016/S0960-9822(99)80420-5 Occurrence Handle10508581

    Article  PubMed  Google Scholar 

  • A. Smogorzewska T. de Lange (2002) ArticleTitleDifferent telomere damage signaling pathways in human and mouse cells Embo. J. 21 4338–4348 Occurrence Handle10.1093/emboj/cdf433 Occurrence Handle12169636

    Article  PubMed  Google Scholar 

  • H. Vaziri S. Benchimol (1998) ArticleTitleReconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span Curr. Biol. 8 279–282 Occurrence Handle10.1016/S0960-9822(98)70109-5 Occurrence Handle9501072

    Article  PubMed  Google Scholar 

  • W.E. Wright J.W. Shay (1992) ArticleTitleThe two-stage mechanism controlling cellular senescence and immortalization Exp. Gerontol. 27 383–389 Occurrence Handle10.1016/0531-5565(92)90069-C Occurrence Handle1333985

    Article  PubMed  Google Scholar 

  • J. Yang E. Chang A.M. Cherry C.D. Bangs Y. Oei A. Bodnar A. Bronstein C.P. Chiu G.S. Herron (1999) ArticleTitleHuman endothelial cell life extension by telomerase expression J. Biol. Chem. 274 26141–26148 Occurrence Handle10.1074/jbc.274.37.26141 Occurrence Handle10473565

    Article  PubMed  Google Scholar 

  • K. Yudoh H. Matsuno F. Nakazawa R. Katayama T. Kimura (2001) ArticleTitleReconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblasts J. Bone Miner. Res. 16 1453–1464 Occurrence Handle11499868

    PubMed  Google Scholar 

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  1. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE, USA, 68198

    Kwang M. Lee, Kyung H. Choi & Michel M. Ouellette

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  1. Kwang M. Lee
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  2. Kyung H. Choi
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  3. Michel M. Ouellette
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Correspondence to Michel M. Ouellette.

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Lee, K.M., Choi, K.H. & Ouellette, M.M. Use of exogenous hTERT to immortalize primary human cells . Cytotechnology 45, 33–38 (2004). https://doi.org/10.1007/10.1007/s10616-004-5123-3

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