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
Full Text
The Full Text of this article is available as a PDF (176.1 KB).
Glossary
- hTERT
human Telomerase Reverse Transcriptase
- hTR -
human Telomerase RNA.
References
- Allsopp R.C., Harley C.B. Evidence for a critical telomere length in senescent human fibroblasts. Exp. Cell Res. 1995;219:130–136. doi: 10.1006/excr.1995.1213. [DOI] [PubMed] [Google Scholar]
- Blackburn E.H. Telomeres: no end in sight. Cell. 1994;77:621–623. doi: 10.1016/0092-8674(94)90046-9. [DOI] [PubMed] [Google Scholar]
- 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. Extension of life-span by introduction of telomerase into normal human cells. Science. 1998;279:349–352. doi: 10.1126/science.279.5349.349. [DOI] [PubMed] [Google Scholar]
- Brenner A.J., Stampfer M.R., Aldaz C.M. Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation. Oncogene. 1998;17:199–205. doi: 10.1038/sj.onc.1201919. [DOI] [PubMed] [Google Scholar]
- Condon J., Yin S., Mayhew B., Word R.A., Wright W.E., Shay J.W., Rainey W.E. Telomerase immortalization of human myometrial cells . Biol. Reprod. 2002;67:506–514. doi: 10.1095/biolreprod67.2.506. [DOI] [PubMed] [Google Scholar]
- Counter C.M., Avilion A.A., LeFeuvre C.E., Stewart N.G., Greider C.W., Harley C.B., Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. Embo. J. 1992;11:1921–1929. doi: 10.1002/j.1460-2075.1992.tb05245.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Counter C.M., Botelho F.M., Wang P., Harley C.B., Bacchetti S. Stabilization of short telomeres and telomerase activity accompany immortalization of epstein-barr virus-transformed human b lymphocytes. J. Virol. 1994;68:3410–3414. doi: 10.1128/jvi.68.5.3410-3414.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Counter C.M., Hahn W.C., Wei W., Caddle S.D., Beijersbergen R.L., Lansdorp P.M., Sedivy J.M., Weinberg R.A. Dissociation among in vitro telomerase activity, telomere maintenanceand cellular immortalization. Proc. Natl. Acad. Sci. USA. 1998;95:14723–14728. doi: 10.1073/pnas.95.25.14723. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Lange T. Protection of mammalian telomeres. Oncogene. 2002;21:532–540. doi: 10.1038/sj.onc.1205080. [DOI] [PubMed] [Google Scholar]
- Di Leonardo A., Linke S.P., Clarkin K., Wahl G.M. DNA damage triggers a prolonged p53-dependent g1 arrest and long-term induction of cip1 in normal human fibroblasts. Gene. Dev. 1994;8:2540–2551. doi: 10.1101/gad.8.21.2540. [DOI] [PubMed] [Google Scholar]
- 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. Genetic and epigenetic changes in human epithelial cells immortalized by telomerase. Am. J. Pathol. 2000;156:1537–1547. doi: 10.1016/S0002-9440(10)65025-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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. The RNA component of human telomerase. Science. 1995;269:1236–1241. doi: 10.1126/science.7544491. [DOI] [PubMed] [Google Scholar]
- Harrington L., Zhou W., McPhail T., Oulton R., Yeung D.S., Mar V., Bass M.B., Robinson M.O., et al. Human telomerase contains evolutionarily conserved catalytic and structural subunits. Gene. Dev. 1997;11:3109–3115. doi: 10.1101/gad.11.23.3109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hayflick L., Moorhead P.S. The serial cultivation of human diploid cell strains. Exp. Cell Res. 1961;25:585–621. doi: 10.1016/0014-4827(61)90192-6. [DOI] [PubMed] [Google Scholar]
- Herbert B.S., Wright W.E., Shay J.W. P16(ink4a) inactivation is not required to immortalize human mammary epithelial cells. Oncogene. 2002;21:7897–7900. doi: 10.1038/sj.onc.1205902. [DOI] [PubMed] [Google Scholar]
- 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. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat. Genet. 1999;21:111–114. doi: 10.1038/5056. [DOI] [PubMed] [Google Scholar]
- 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. Isolation of a candidate human telomerase catalytic subunit genewhich reveals complex splicing patterns in different cell types. Hum. Mol. Genet. 1997;6:2011–2019. doi: 10.1093/hmg/6.12.2011. [DOI] [PubMed] [Google Scholar]
- 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. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994;266:2011–2015. doi: 10.1126/science.7605428. [DOI] [PubMed] [Google Scholar]
- Kiyono T., Foster S.A., Koop J.I., McDougall J.K., Galloway D.A., Klingelhutz A.J. Both pRB/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells . Nature. 1998;396:84–88. doi: 10.1038/23962. [DOI] [PubMed] [Google Scholar]
- Lee K.M., Nguyen C., Ulrich A.B., Pour P.M., Ouellette M.M. Immortalization with telomerase of the nestin-positive cells of the human pancreas. Biochem. Biophys. Res. Commun. 2003;301:1038–1044. doi: 10.1016/S0006-291X(03)00086-X. [DOI] [PubMed] [Google Scholar]
- 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. Telomerase maintains telomere structure in normal human cells. Cell. 2003;114:241–253. doi: 10.1016/S0092-8674(03)00550-6. [DOI] [PubMed] [Google Scholar]
- 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. Hest2, the putative human telomerase catalytic subunit geneis up-regulated in tumor cells and during immortalization. Cell. 1997;90:785–795. doi: 10.1016/s0092-8674(00)80538-3. [DOI] [PubMed] [Google Scholar]
- 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. Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat. Genet. 1999;21:115–118. doi: 10.1038/5063. [DOI] [PubMed] [Google Scholar]
- Morales C.P., Gandia K.G., Ramirez R.D., Wright W.E., Shay J.W., Spechler S.J. Characterisation of telomerase immortalised normal human oesophageal squamous cells. Gut. 2003;52:327–333. doi: 10.1136/gut.52.3.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakamura T.M., Morin G.B., Chapman K.B., Weinrich S.L., Andrews W.H., Lingner J., Harley C.B., Cech T.R. Telomerase catalytic subunit homologs from fission yeast and human. Science. 1997;277:955–959. doi: 10.1126/science.277.5328.955. [DOI] [PubMed] [Google Scholar]
- Ouellette M.M., McDaniel L.D., Wright W.E., Shay J.W., Schultz R.A. The establishment of telomerase-immortalized cell lines representing human chromosome instability syndromes. Hum. Mol. Genet. 2000;9:403–411. doi: 10.1093/hmg/9.3.403. [DOI] [PubMed] [Google Scholar]
- Ouellette M.M., Lee K. Telomerase: diagnostics, cancer therapeutics and tissue engineering. Drug Discov. Today. 2001;6:1231–1237. doi: 10.1016/S1359-6446(01)02052-9. [DOI] [PubMed] [Google Scholar]
- Ramirez R.D., Morales C.P., Herbert B.S., Rohde J.M., Passons C., Shay J.W., Wright W.E. Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions. Gene. Dev. 2001;15:398–403. doi: 10.1101/gad.859201. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robles S.J., Adami G.R. Agents that cause DNA double strand breaks lead to p16ink4a enrichment and the premature senescence of normal fibroblasts. Oncogene. 1998;16:1113–1123. doi: 10.1038/sj.onc.1201862. [DOI] [PubMed] [Google Scholar]
- Romanov S.R., Kozakiewicz B.K., Holst C.R., Stampfer M.R., Haupt L.M., Tlsty T.D. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature. 2001;409:633–637. doi: 10.1038/35054579. [DOI] [PubMed] [Google Scholar]
- Shay J.W., Pereira-Smith O.M., Wright W.E. A role for both rb and p53 in the regulation of human cellular senescence. Exp. Cell Res. 1991;196:33–39. doi: 10.1016/0014-4827(91)90453-2. [DOI] [PubMed] [Google Scholar]
- Shay J.W., West M.D., Wright W.E. Re-expression of senescent markers in deinduced reversibly immortalized cells. Exp. Gerontol. 1992;27:477–492. doi: 10.1016/0531-5565(92)90003-I. [DOI] [PubMed] [Google Scholar]
- Shay J.W., Der Haegen B.A., Ying Y., Wright W.E. The frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with sv40 large t-antigen. Exp. Cell Res. 1993;209:45–52. doi: 10.1006/excr.1993.1283. [DOI] [PubMed] [Google Scholar]
- Shay J.W., Wright W.E. Telomerase activity in human cancer. Curr. Opin. Oncol. 1996;8:66–71. doi: 10.1097/00001622-199601000-00012. [DOI] [PubMed] [Google Scholar]
- Shay J.W., Bacchetti S. A survey of telomerase activity in human cancer. Eur. J. Cancer. 1997;33:787–791. doi: 10.1016/S0959-8049(97)00062-2. [DOI] [PubMed] [Google Scholar]
- Shelton D.N., Chang E., Whittier P.S., Choi D., Funk W.D. Microarray analysis of replicative senescence. Curr. Biol. 1999;9:939–945. doi: 10.1016/S0960-9822(99)80420-5. [DOI] [PubMed] [Google Scholar]
- Smogorzewska A., de Lange T. Different telomere damage signaling pathways in human and mouse cells. Embo. J. 2002;21:4338–4348. doi: 10.1093/emboj/cdf433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vaziri H., Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr. Biol. 1998;8:279–282. doi: 10.1016/S0960-9822(98)70109-5. [DOI] [PubMed] [Google Scholar]
- Wright W.E., Shay J.W. The two-stage mechanism controlling cellular senescence and immortalization. Exp. Gerontol. 1992;27:383–389. doi: 10.1016/0531-5565(92)90069-C. [DOI] [PubMed] [Google Scholar]
- Yang J., Chang E., Cherry A.M., Bangs C.D., Oei Y., Bodnar A., Bronstein A., Chiu C.P., Herron G.S. Human endothelial cell life extension by telomerase expression. J. Biol. Chem. 1999;274:26141–26148. doi: 10.1074/jbc.274.37.26141. [DOI] [PubMed] [Google Scholar]
- Yudoh K., Matsuno H., Nakazawa F., Katayama R., Kimura T. Reconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblasts. J. Bone Miner. Res. 2001;16:1453–1464. doi: 10.1359/jbmr.2001.16.8.1453. [DOI] [PubMed] [Google Scholar]