[Cell Cycle 5:5, 465-466, 1 March 2006]; ©2006 Landes Bioscience
Telomerase Expression in Somatic Cells Cutting Edge View
Fountain of Youth or Damocles’ Sword? Normal somatic cells have a limited proliferative potential, strictly linked to their insufficient telomerase expression and to the consequent telomere shortening.1 When normal cells grown in culture expire the number of divisions allowed, they stop replicating and enter a phase known as “cellular senescence”.2 Restoration of telomerase activity stabilizes telomeres and allows many types of somatic cells to bypass senescence and to become immortal. Induction of telomerase activity was proposed as a mean to rejuvenate tissues for therapeutic purposes, reestablishing cellular functions.3 However, an artificial extension of cellular lifespan could come at a price, increasing the probability of neoplastic transformation. The ability to divide indefinitely is a typical characteristic of tumor cells and is associated with the accumulation of genetic and epigenetic changes, which can contribute to the malignant phenotype and aggressiveness of tumors. If normal cells do not have any constraint to replicate, as is the case in cellular senescence, they can accumulate mutations and undergo neoplastic transformation. Although many examples support the notion that telomerase immortalized cells maintain a normal phenotype for several population doublings, a growing body of evidence suggests that telomerase immortalized cells can acquire cancer associated changes (ref. 4 and refs. therein). We have immortalized through ectopic telomerase expression skin fibroblasts derived from a centenarian individual and have propagated them continuously.4,5 During culture propagation, these cells, named cen3tel, gradually changed their characteristics, they started growing faster, reaching a greater culture saturation density, and they lost contact growth inhibition and the requirement of serum for growth. At the chromosomal level, cells became first trisomic for a few chromosomes, then acquired clonal structural chromosomal rearrangements and, finally, became subtetraploid. We cannot exclude that the old age of the cell donor contributed to the chromosomal instability of the fibroblast strain. However, chromosomal instability per se was not sufficient to induce transformation in the absence of constitutive telomerase expression, probably because senescence stopped cellular proliferation and thus the accumulation of further rearrangements. Interestingly, we found a similar cytogenetic pattern also in a second fibroblast strain, derived from another centenarian individual that we immortalized by telomerase expression.5 In both immortalized cell lines, marker chromosomes derived from the rearrangement of a trisomic chromosome were observed, suggesting that an abnormal ploidy can drive the formation of structural abnormalities. Successive stepwise mutations in tumor suppressor genes and in oncogenes drove neoplastic transformation of cen3tel cells. Downregulation of the tumor suppressor genes6 p16INK4a and p14ARF was first observed in cen3tel cells, it was associated with the ability of cells to grow in the absence of solid support, but was not sufficient to render the cells tumorigenic. A mutation in the DNA binding domain of p53 and overexpression of c-myc were subsequently detected in cen3tel cells, which became able to form tumors in immunocompromised mice in less than one month. Histological analysis revealed that tumors were fibrosarcomas. Further propagation in culture made cen3tel cells more aggressive; in fact, cells formed tumors within eight days from inoculation and tumors grew faster. These tumors were fibrosarcomas as well, with an increased degree of vascularization as compared to those induced by younger cen3tel cells, which could explain the faster growth (our unpublished results). Interesting features of tumorigenic cen3tel cells were the loss of fibroblastic morphology and acquisition of a polygonal shape,4 a morphological transition opposite to the epithelial-mesenchymal transition observed in tumor cells of epithelial origin.7 It will be of interest to determine whether these morphological changes have a causal role in neoplastic transformation of fibroblasts or are just a consequence. Loss of p16INK4a and mutation in p53 were not isolated findings in telomerase immortalized cen3tel cells. In fact, in a cell sample propagated separately from the main cellular population, these genetic changes were also detected; p53 was mutated in a different
*Correspondence to: Chiara Mondello; Istituto di Genetica Molecolare; CNR; Via Abbiategrasso, 207; 27100, Pavia, Italy; Tel.: +39.0382.546332; Fax: +39.0382.422286; Email:
[email protected] Original manuscript submitted: 01/12/06 Manuscript accepted: 01/13/06
KEY WORDS telomeres, telomerase, cellular senescence, transformation, cancer, tumor suppressor genes, oncogenes
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We are grateful to Dr. Eugenio Scanziani of the University of Milan (Italy) for histological and histochemical analysis of tumors induced by cen3tel cells. Work in C.M. lab is supported by the EU grant FIGHT CT 2002 217 and by the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (FIRB RBNE01RNN7 and RBAU01ZB78).
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Previously published as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=2499
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2Istituto di Ricerche Farmacologiche "Mario Negri"; Milan, Italy
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1Istituto di Genetica Molecolare; CNR; Pavia, Italy
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Chiara Mondello1,* Samantha Zongaro1 Maurizio D'Incalci2
www.landesbioscience.com
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Telomerase and Cellular Transformation
codon, indicating that loss of p53 function was an event independent from that which occurred in the main cell line. These cells did not overexpress c-myc and were tumorigenic with a longer latency, suggesting that the acquisition of additional mutations was required to induce tumor formation (our unpublished results). In the cen3tel cellular system, telomerase immortalization has probably been the basis for the acquisition of cancer-associated mutations. Although evidence has been reported that telomerase has additional functions in tumorigenesis besides telomere maintenance, its role in telomere stabilization and in the prolongation of cellular lifespan could be sufficient to allow the selection of cells with mutations relevant for transformation. According to this view, telomerase would not be a classical “oncogene”, but could “free” the way toward transformation; thus, its activation in somatic cells could actually be a Damocles’ sword. Telomerase immortalization of human somatic cells has been exploited to construct human tumor cell lines engineered with defined genetic elements and to identify critical pathways whose experimental disruption leads to the tumorigenic phenotype.8 Telomerase immortalized cell lines, which undergo stepwise transformation, such as cen3tel, are a complementary tool to investigate molecular changes that are spontaneously associated with tumorigenesis. The availability of cell samples in culture at different stages of transformation can allow the identification of genetic determinants specifically linked to tumor initiation and progression. In addition, cell samples with specific tumorigenic determinants can be an instrument to test new therapeutic approaches directed to molecular alterations associated with different phases of malignant transformation. Further Reading 1. 2. 3. 4. 5. 6. 7. 8.
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Greider CW, Blackburn EH. Cell 2004; 116:S83-6, (1 p following S86). Campisi J. Cell 2005; 120:513-22. Bodnar AG, Ouellette M, Frolkis M, et al. Science 1998; 279:349-52. Zongaro S, de Stanchina E, Colombo T, et al. Cancer Res 2005; 65:11411-8. Mondello C, Chiesa M, Rebuzzini P, et al. Biochem Biophys Res Commun 2003; 308:914-21. Voorhoeve PM, Agami R. Cell Cycle 2004; 3:616-20. Thiery JP. Nat Rev Cancer 2002; 2:442-54. Boehm JS, Hahn WC. Curr Opin Genet Dev 2005; 15:13-7.
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