Telomerase: cellular immortalization and neoplastic ... - CiteSeerX

Telomerase and Alternative Lengthening of Telomeres Cytogenet Genome Res 122:255–262 (2008) DOI: 10.1159/000167811

Telomerase: cellular immortalization and neoplastic transformation. Multiple functions of a multifaceted complex C. Belgiovine I. Chiodi C. Mondello Istituto di Genetica Molecolare, CNR, Pavia (Italy) Accepted in revised form for publication by P. Slijepcevic, 18 July 2008.

Abstract. The telomerase complex allows telomere length maintenance, which is required for an unlimited cellular proliferation. Telomerase is virtually absent in normal human somatic cells, which are characterized by a definite proliferation potential, while it is present in the vast majority of tumors (around 90%). Restoring telomerase activity in normal somatic cells can indefinitely prolong cellular life span. However, evidence has been reported that this event can be associated with the acquisition of characteristics typ-

ical of cellular transformation. Moreover, analysis of telomerase immortalized cells, as well as of tumor cells in which telomerase is inactivated, has highlighted multiple functions of telomerase in tumorigenesis, besides telomere lengthening. In this paper, we will review telomerase immortalization of somatic cells, together with its possible consequences, and we will examine the complex role of telomerase in tumorigenesis.

In eukaryotes, chromosomes end with specialized structures, the telomeres. Telomeres are essential for the maintenance of genome integrity: they guarantee the complete replication of linear genomes, protect chromosome ends from degradation, and assure chromosomal stability (Blackburn et al., 2006). Telomeres are composed of repetitions of short motifs, in humans the hexanucleotide TTAGGG, bound to specific proteins and are extended at each cell cycle by the specialized reverse transcriptase enzyme telomerase. Several subunits participate in the formation of the telomerase complex, but the main components that co-purify with the catalytic activity are: the reverse transcriptase catalytic subunit (TERT: Telomerase Reverse Transcriptase; hTERT: hu-

man TERT), the RNA molecule (TERC: Telomerase RNA Component), which contains the template for the synthesis of the telomeric repeats, and dyskerin (Collins, 2006; Cohen et al., 2007). The maintenance of telomere length within a range specific for each species (in humans about 6–20 kb) is crucial for the proper functioning of telomeres, together with their correct binding to telomeric proteins (de Lange, 2005). In humans, telomeres shorten at each cell division (Harley, 1991) because of the low level of telomerase, which is detected only during the S-phase and is insufficient to elongate telomeres (Masutomi et al., 2003). Telomere shortening limits the proliferative potential of somatic cells; in fact, telomeres that fall below a threshold length are not recognized as the ends of the chromosomes anymore, but as DNA double strand breaks that trigger a DNA damage response (DDR) (d’Adda di Fagagna et al., 2003). DDR leads cells to arrest proliferation and enter a phase known as replicative senescence (Campisi and d’Adda di Fagagna, 2007). If mutations in DDR genes and in genes controlling proliferation occur, cells can extend their replicative potential. Inactivation of the TP53 and RB1/p16INK4a tumor suppressor pathways is a key event for the bypass of senescence (Shay et al., 1991). However, in the absence of telomere maintenance

Work in the C.M. laboratory is supported by Fondazione Cariplo (Grant 20060734). C.B. is a PhD student of the University of Pavia (Dottorato in Scienze Genetiche e Biomolecolari). I.C. post-doctoral fellowship is supported by Fondazione Cariplo. Request reprints from Chiara Mondello Istituto di Genetica Molecolare, CNR via Abbiategrasso 207, Pavia (Italy) telephone: +39 0382 546332; fax: +39 0382 422286 e-mail: mondello@igm.cnr.it

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mechanisms, telomeres of cells that have circumvented senescence keep on shortening at every cell cycle, leading to massive chromosomal instability and cell death (Counter et al., 1992). In this view, telomere shortening has been sought as a tumor suppressor mechanism, which does not allow cells to replicate indefinitely when carrying mutations threatening genome integrity. Only cells that can counteract telomere shortening can proliferate indefinitely; this is the case for tumor cells, which, in the vast majority of the cases (around 90%), present telomerase activity (Harley, 2008), and in the others show the activation of alternative telomere length maintaining mechanisms (ALT) based on recombination (Muntoni and Reddel, 2005). The strict relationship between telomere shortening and replicative senescence, on the one hand, and telomerase expression and cellular immortality, on the other hand, was demonstrated when the gene coding for the telomerase catalytic subunit was cloned and shown to confer indefinite proliferation potential when introduced into several types of somatic cells (Bodnar et al., 1998). Telomerase immortalization of human somatic cells

In human somatic cells in culture, the expression of the telomerase catalytic subunit gene is the limiting event to have telomerase activity; in fact, introduction of the cDNA for hTERT is sufficient to restore telomerase activity (Bodnar et al., 1998). A sure effect of telomerase reactivation is telomere stabilization. In most cases, telomeres are lengthened, in other cases they keep on shortening before stabilization; however, even if their length stabilizes below the critical measure associated with senescence, in the presence of telomerase activity, they remain functional and do not lead to the formation of telomeric fusions or to replication arrest (Ouellette et al., 1999; Zongaro et al., 2008). This observation supports the hypothesis that telomerase plays a role in telomere maintenance that goes beyond telomere lengthening. Telomerase reactivation is associated with the prolongation of cellular life span. The initial extension of the proliferative capacity is mainly achieved without the requirement of additional genetic changes (for a review see Harley, 2002); nevertheless, a common finding in telomerase immortalized cells is the loss of expression of p16INK4a (a transcript variant of the CDKN2A gene). In human keratinocytes, loss of p16INK4a expression is a stringent requirement for immortalization. In fact, telomerase alone fails to immortalize this type of cells if CDKN2A (p16INK4a ) is not inactivated (Kiyono et al., 1998). It was suggested that this requisite is due to cell culture conditions which, stressing the cells, induce p16INK4a expression and the replicative block. Evidence has been reported that some epithelial cells can be immortalized by hTERT alone when cultured in the presence of feeder cells, and not directly on plastic (Ramirez et al., 2001; Herbert et al., 2002; Harada et al., 2003). However, Darbro et al. (2006) showed that CDKN2A (p16INK4a ) promoter undergoes methylation, becoming inactive, also in human ke-

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Cytogenet Genome Res 122:255–262 (2008)

ratinocytes co-cultured with feeder cells. These authors were unable to obtain immortal cells from hTERT transduced keratinocytes grown on plastic, but managed to do so when cells were grown on a feeder layer and then transferred on plastic. These cells showed CDKN2A (p16INK4a) promoter methylation, suggesting that the extension of the proliferative capacity on feeder cells allowed the genesis of the epigenetic alterations associated with CDKN2A downregulation. A less stringent requirement for p16INK4a loss of expression seems to characterize telomerase immortalization of human fibroblasts. Several examples of telomerase immortalized fibroblast strains have been described in which no additional genetic changes were detected after several passages in culture (Harley, 2002). Nevertheless, in as many cases, CDKN2A (p16INK4a ) downregulation was found after hTERT introduction, suggesting that other barriers to immortalization can be present at least in some fibroblast strains. In MRC5 fibroblast clones obtained after hTERT transduction, for example, Taylor et al. (2004) described the occurrence of a telomere independent crisis during immortalization, which was overcome by those clones that had lost p16INK4a expression. Telomerase has been ectopically expressed in several types of human somatic cells from different tissues in the attempt to immortalize them (see Harley, 2002 and Table 1). For many cell types immortalization has been achieved; however, in several instances additional genetic modification had to be introduced to gain immortal cell lines, or genetic modifications were acquired by the cells during the extension of the life span. For some cell types, contradictory results were obtained in different laboratories. As far as T lymphocytes are concerned, for example, Migliaccio et al. (2000) did not even manage to extend cellular life span by ectopic telomerase expression, while Rufer et al. (2001) and Hooijberg et al. (2000) succeeded in getting a dramatic extension of the proliferative capacity. Similarly Yang et al. (2007a, b) could immortalize ovarian surface epithelial cells by hTERT expression only simultaneously inactivating either TP53 or RB1, whereas Li et al. (2007) obtained immortalized cell lines by the sole induction of telomerase activity. These different results could reflect genetic heterogeneity among the cells used in the diverse experiments, or even differences in cell culture conditions. Taken together, the observations presented so far highlight the complexity of life span regulation in somatic human cells and indicate that telomerase is essential to get cellular immortalization, but it might not be sufficient. Telomerase immortalization and cellular transformation

Besides CDKN2A (p16INK4a ) inactivation, a large body of evidence shows that long term propagation in culture of telomerase immortalized cells is frequently associated with the activation of cancer pathways, such as, overexpression

Table 1. Ectopic hTERT expression in human somatic cells Cell type

Tissue

Immortalizationa

Genetic Specific featuresc changesb

Epithelial

Adenoid

+ (only after crisis)

+

Epithelial (keratinocytes) Epithelial

Oesophagous

+

–

Bronchus

+

Epithelial Epithelial

Cornea Airway

– (Cdk4 overexpression) + +


Urothelium Nasopharynx

+ +

– +


Ovarian surface Ovarian surface

– (inactivation of pRb) + – (inactivation of p53) +


+ +

– +

+ + + – (HPV E6/E7) +

– – – – + +

Endothelial

Ovarian surface Fetal liver hepatocytes Prostate Muscle Embryo Fetal spinal cord Cord blood Large & micro vessels Umbical vein

+

–

T Lymphocytes T Lymphocytes T Lymphocytes Trophoblasts

Blood Blood Blood Placenta

+/– +/– +

– – – –

Epithelial Stem cells Stem cells Stem cells Stem cells Endothelial

+ +

Downregulation of p16INK4a or p14ARF, chromosome aberrations, defective differentiation Moderate basal cell hyperplasia in organotypic culture, no transformation Duplication of a part of chromosome 5 and 20 Loss of p16INK4a expression, no transformation Downregulation of CDKN2A (p16INK4a) at late passages – Deletion of CDKN2A, downregulation ofRASSF1A, duplication 17q21–q25, activation of Nf-KB Altered cell cycle proteins Altered cell cycle proteins, reduced response to ␥-ray – Karyotype anomalies, no transformation Expression of stem cell markers Extended lifespan Able to differentiate Able to differentiate Minimal chromosomal aberrations Differentiated phenotype, form microvascular structures Differentiated phenotype, particular expression profile No extended lifespan Extended life span Extended life span Normal cell properties

References

Farwell et al., 2000 Harada et al., 2003 Ramirez et al., 2004 Robertson et al., 2005 Piao et al., 2005 Chapman et al., 2006 Li et al., 2006 Yang et al., 2007a Yang et al., 2007b Li et al., 2007 Haker et al., 2007 Li et al., 2008 Di Donna et al., 2003 Xu et al., 2004 Roy et al., 2004 Akimov et al., 2005 Yang et al., 1999, 2001 Chang et al., 2005 Migliaccio et al., 2000 Hooijberg et al., 2000 Rufer et al., 2001 Wang et al., 2006

a

+: immortalization was achieved by hTERT expression alone; –: immortalization required additional exogenous genetic modifications; modifications are reported in parenthesis. b +: cells acquired genetic modification during prolongation of life span. c Cell line characteristics and genetic modifications acquired during propagation in vitro.

of the proto-oncogenes MYC and BMI1 (Wang et al., 2000; Milyavsky et al., 2003), resistance to growth inhibition induced by transforming growth factor ␤ (Stampfer et al., 2001), loss of p53 function (Noble et al., 2004) and p14ARF expression (p14ARF is another splice variant of CDKN2A) (Milyavsky et al., 2003). Despite these genetic alterations, these cells did not display tumorigenicity in immunocompromised mice, which, in contrast, was detected both in telomerase immortalized adult mesenchymal stem cells (HMSC) (Serakinci et al., 2004) and in human TERT immortalized fibroblasts in our laboratory (Zongaro et al., 2005). In two independent hTERT transduced HMSC cell lines deletion of the CDKN2A locus was found and the acquisition of the tumorigenic potential was associated with an inactivating mutation in K-ras or in the tumor suppressor gene DBC1, respectively (Serakinci et al., 2004). Analysis

of clonal populations derived from the DBC1 mutated cell line revealed heterogeneity in the population, in fact, while four clones were fully tumorigenic, such as the parental population, one of them developed latent tumors, and the last one developed tumors with a 30% penetrance (Burns et al., 2005). Interestingly, poor tumorigenicity correlated with high levels of CDKN1B (p27kip1) expression, while high tumorigenicity correlated with high levels of cyclin D1 expression. In our laboratory we transduced with hTERT cDNA a human fibroblast cell line derived from a centenarian individual and derived an immortal cell line (cen3tel) that undertook neoplastic transformation (Mondello et al., 2003; Zongaro et al., 2005). During the first period of propagation in culture, cen3tel cells behaved as parental fibroblasts, then showed numerical and structural chromosomal aberra-


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tions. Subsequently they acquired the ability to grow in the absence of solid support, a characteristic which was paralleled by downregulation of CDKN2A, with loss of detectable p16INK4a protein. Finally, after further propagation in culture, the cells displayed tumorigenic ability, leading to tumor development when subcutaneously injected into immunocompromised mice. Tumorigenic cells were subtetraploid, had lost TP53 expression because of a mutation in the DNA binding domain of the protein, and showed overexpression of the MYC gene. Interestingly, we also observed the occurrence of such genetic alterations in a population derived from the original hTERT transduced cells and propagated in culture independently from cen3tel cells. This second cell line, named cen3tel S2, became tumorigenic in immunocompromised mice as well. It showed loss of p16INK4a expression, TP53 mutation and MYC overexpression (our unpublished results). The first two events occurred independently from those observed in cen3tel cells; in fact, while in cen3tel cells the absence of p16INK4a was due to the lack of its transcript, in cen3tel S2 cells, the protein was undetectable but the RNA was still present; TP53 did not show the mutation in the DNA binding motif, but was mutated in the tetramerization domain. Taken together, the results obtained with cen3tel and cen3tel S2 cells indicate that mutations in CDKN2A, TP53 and MYC play a fundamental role in neoplastic transformation of human fibroblasts. Does hTERT expression and/or telomerase activity have a direct role in neoplastic transformation of the immortalized cells? From the data collected on the cells described above, it is impossible to draw any conclusion about this point. In fact, the genetic changes observed occurred after several passages since the induction of telomerase expression, suggesting that the artificial prolongation of cellular life span can give the cells time to acquire the mutations linked to neoplastic transformation. Since these mutations confer a selective advantage to the cells grown in vitro, mutated cells can easily overtake the population. However, other evidence suggests that telomerase itself can modulate gene expression and through this way it can directly influence cellular growth. Smith et al. (2003) observed that mammary epithelial cells ectopically expressing telomerase show a less stringent requirement for mitogens to proliferate compared to control cells. When these authors performed microarray analysis on mammary epithelial cells at early passages after transduction with hTERT, they found that the increased proliferation correlated with the overexpression of several genes that have a stimulatory effect on cellular proliferation, suggesting that telomerase activation is accompanied by induction of a cellular mitogenic program. One of the genes induced was the epidermal growth factor receptor (EGFR) gene, which stimulates mammary epithelial cell growth and is involved in breast cancer tumorigenesis. Inhibition of EGFR expression reverted the increased proliferation ability of telomerase expressing cells. Interestingly, this effect of hTERT expression apparently depends on the restoration of telomerase enzymatic activity, since transduction with a gene coding for a catalytically inactive hTERT does not change the growth char-

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acteristics of the cells and does not induce the expression of EGFR. Increased proliferation was also observed in human fibroblasts ectopically expressing hTERT. Lindvall et al. (2003) found that one of the genes upregulated in different fibroblast strains transduced with hTERT is epiregulin (EREG), a potent growth factor belonging to the EGF family. Again, blockade of epiregulin function with a neutralizing antibody reduced the growth capability of transduced cells. Protein profiling of hTERT immortalized retinal pigment epithelium (RPE) cells revealed clear changes in protein expression compared to primary RPE cells (Alge et al., 2006). In particular, the main changes can be attributed to an increase in adhesional strength, cell contraction, cellmatrix interactions, extracellular matrix deposition and modification, as well as loss of polarization, variations pointing to a dedifferentiation of telomerase immortalized cells. All together the observations presented above show that ectopic telomerase expression can modify the expression profile of immortalized cells and that modifications can vary depending on cell type. In different cell types, modifications involve genes that take part in tumorigenesis, indicating that telomerase can play additional functions beyond telomere maintenance during tumor development. Telomerase immortalization has been viewed as a possible means to treat human diseases and age-related disorders and as a way to obtain large amounts of cells to be used in regenerative medicine (Petersen and Niklason, 2007); however, the results obtained so far suggest that caution should be taken in the use of telomerase immortalized cells for therapeutic purposes, because of the frequent appearance of mutations in cancer genes in immortalized cells. For a medical application of in vitro immortalized cells, further studies should be undertaken to minimize the chance that cells undergo cancer associated mutations and great attention should be paid in controlling the status of cancer associated genes before immortalized cells are introduced in a living organism. The use of safer vectors for the introduction of hTERT, such as lentiviral vector (Bocker et al., 2008), could also be of help in decreasing instability of immortalized cells. Telomerase, a key player for the experimental creation of models for human tumors

Telomerase immortalized cell lines that undergo spontaneous transformation are a useful tool to underscore genomic variations accompanying tumorigenesis. In a parallel approach, telomerase immortalization has been exploited to create tumor cell lines by transformation with defined viral and/or cellular oncogenes. In contrast to mouse cells, human somatic cells challenged with several combinations of oncogenes do not undergo transformation. One very important difference between human and rodent cells concerns telomere biology.

In fact, mouse cells have much longer telomeres, frequently undergo spontaneous transformation, and often show telomerase activity (Sherr and DePinho, 2000); in contrast, human cells undergo senescence when they exhaust the telomeric DNA reservoir, do not acquire telomerase activity and do not transform spontaneously. The possibility to bypass senescence through ectopic telomerase expression has allowed transformation of human cells with defined genetic elements. In initial experiments, it was demonstrated that normal human fibroblasts and epithelial cells can be converted into tumorigenic cells by the sequential introduction of hTERT, SV40 early region and oncogenic ras (Hahn et al., 1999). Thus, although it cannot be excluded that clonal selection contributed to the isolation of malignant cells with unidentified mutations (Mahale et al., 2008), these experiments indicated that conversion of normal cells into malignant ones requires a minimum set of genetic changes, namely, activation of telomere maintenance, inactivation of TP53 and RB1 pathways, by SV40 large T, perturbation of the serine-threonine protein phosphatase 2A, by SV40 small t (both large T and small t are encoded by the SV40 early region) (Hahn et al., 2002), and constitutional activity of the ras pathway. So far, several types of primary human cells have been transformed with a combination of defined genetic elements giving rise to experimental models closely resembling specific types of cancers (Kendall et al., 2005; Schinzel and Hahn, 2008). Seger et al. (2002) managed to convert primary fibroblasts into cancer cells by combining expression of adenovirus E1A, oncogenic H-rasV12 and MDM2 without induction of hTERT expression. Genetically modified cells were able to generate tumors in immunocompromised mice, however tumor cells were not immortal. In fact, tumor cells explanted into culture underwent telomere shortening and crisis; clonal populations emerging from crisis showed telomerase activity. Hence, this particular combination of oncogenes seems to be sufficient to transform cells in the absence of telomerase activity; however, even in this case, to get immortal tumor cells, and thus fully tumorigenic conversion and tumor progression, telomerase activity is absolutely required. Telomerase activities besides telomere lengthening

Nowadays, there is more and more evidence that telomerase plays a role in tumorigenesis that goes beyond telomere length maintenance. As previously mentioned, the maintenance of telomere length is achieved by expressing telomerase or through the ALT mechanism. Whereas more than 90% of human cancers express telomerase, only 10% maintain their telomeres through ALT, suggesting that the two mechanisms are not equivalent during tumor formation. In support of this hypothesis, Stewart et al. (2002) showed that the immortal human fibroblast cell line GM847, which is transformed with H-RasV12 oncoprotein, lacks telomerase activity and exhibits the ALT phenotype, did not

form tumors in immunodeficient nude mice, whereas it became tumorigenic after hTERT expression. Interestingly, telomerase activity and ALT coexisted in these cells, as proven by the presence of ALT-associated PML bodies, a marker of the ALT phenotype (Perrem et al., 2001). Tumorigenicity was also observed in GM847 cells transfected with HA tagged hTERT, a chimeric protein able to elongate telomeres in vitro but not in vivo (Stewart et al., 2002). These data clearly indicate that, in this experimental system, telomere elongation by telomerase is not critical for tumor development and hTERT gives a contribution to tumorigenesis other than telomere lengthening. Evidence for non-telomeric effects of telomerase in tumor development was also obtained in mouse models. Overexpression of mTERT (mouse TERT) in FVB/N mice enormously augmented tumorigenesis over the lifetime of the animals (Artandi et al., 2002). Significantly, in most cases transgenic mice developed breast cancer, despite the resistance of the FBV strain to this type of tumor and the broad range of tissues in which the transgene was expressed, suggesting that the telomerase-induced cancer-prone phenotype is tissue specific. These results reveal, first of all, that TERT promotes cancer even in the presence of ample telomere reserves, as in the mouse cells, indicating functions beyond its ability to sustain telomere length, and that telomerase influences spontaneous tumor susceptibility. Increased tumor induction upon exposure to chemical carcinogens was described in mice ectopically expressing the catalytic subunit of telomerase in basal keratinocytes (K5-mTERT mice) (Gonzalez-Suarez et al., 2001). In these mice, ectopic mTERT expression did not result in an extension of telomeres, but was associated with an increased susceptibility in developing papillomas after exposure to chemical carcinogens. Because an increased proliferation of basal keratinocytes was observed after treatment of K5-mTERT mice with chemical carcinogens, as well as a faster rate of re-epithelisation of the skin in wound healing experiments, it was proposed that ectopic telomerase expression conferred a proliferative advantage, which could be involved in promoting tumor formation and progression. Cayuela et al. (2005) demonstrated that both mTERT and mTERC are required for the increased tumorigenesis. In fact, K5-mTERT mice in which TERC had been knocked down were not prone to tumor development. Surprisingly, TERT overexpression in the absence of TERC had a protective role in relation to tumor formation. Overexpression of a mutant template human TERC in telomerase-positive cancer cells, as well as expression of a short-hairpin interfering RNA against hTERC, inhibited cell growth and induced apoptosis very rapidly, without telomere shortening (Li et al., 2004). A similarly rapid growth inhibitory and apoptotic response was observed in cancer cells in which hTERT expression had been knocked down by antisense expression (Folini et al., 2003), indicating that the reduction of the telomerase ribonuclear complex levels has a dramatic effect on cancer cell viability independently of telomerase telomeric effects. Li et al. (2005) showed that the quick response to TERC depletion was re-


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lated to a modulation of gene expression, with a decreased transcription of genes involved in cell cycle progression, tumor growth, angiogenesis and metastasis. Recently, several studies have assigned a role to telomerase in the regulation of cellular metabolic pathways, such as glycolysis. Cancer cells frequently display high rates of aerobic glycolysis in comparison to non-transformed ones, a phenomenon known as the Warburg effect (Warburg, 1956), and an increased glucose uptake is a biochemical marker of cancer. Bagheri et al. (2006) found that telomerase RNA knockdown in a model of murine melanoma metastasis reduced telomere length, tumor invasion and metastatic potential. Interestingly, these authors showed that telomerase targeting was followed by the downregulation of genes involved in glycolytic pathways, such as aldolase C and phosphofructokinase, indicating that telomerase can alter the energy state of tumor cells. Moreover, hTERT is an AKT kinase substrate protein (Kang et al., 1999). AKT is a serine/threonine kinase activated in cancer cells, which exerts a direct influence on glucose metabolism rendering cancer cells dependent on aerobic glycolysis (Elstrom et al., 2004). Since treatment with a specific Akt inhibitor downregulates hTERT phosphorylation and then telomerase activity (Kang et al., 1999), it is possible to suppose that telomerase can play a major role as an intermediary between Akt and the glycolytic pathway. Sharma et al. (2003) obtained evidence that telomerase can regulate gene expression by altering telomere interactions with the nuclear matrix (Smith and de Lange, 1997). In human fibroblasts ectopically expressing telomerase, they showed a strict relationship between binding of telomerase to the telomeric DNA, changes in the interactions of telomeres with the nuclear matrix and transcriptional modification of a subset of genes. In particular, in cells overexpressing telomerase there was an upregulation of genes involved in chromatin modification and DNA damage repair processes. Consistently, cells showed a rate of DNA repair higher than wild type cells and an enhanced genomic stability. The timing of these phenomena, i.e. hTERT telomere association and alterations in gene expression, suggests that a change in telomeric chromatin organization may influence the pattern of transcription. Once again, telomerase activity at telomeres did not mediate these functions. Telomerase expression seems to modulate chromatin state not only at telomeric regions, but also throughout the genome. Trichostatin A (TSA), a histone deacetylase inhibitor, induces changes in chromatin organization, rapidly leading to ATM autophosphorylation. In human primary fibroblasts, knockdown of hTERT impaired TSA induced ATM phosphorylation, suggesting that suppression of hTERT expression modulates chromatin organization (Masutomi et al., 2005). Moreover, chromatin prepared from hTERT lacking cells was slightly more susceptible to micrococcal nuclease digestion and showed decreased levels of H3-lysine 9 dimethylation and H4-lysine 12 acetylation, together with increased amounts of H3-lysine 9 acetylation. In addition, cells lacking hTERT showed variations in H2AX solubility, together with impaired DNA damage responses,

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including H2AX phosphorylation, increased sensitivity to ionizing radiations and reduced repair of DNA double strand breaks. The hTERT chromatin effects described by Masutomi et al. (2005) suggest a possible mechanism by which hTERT regulates DNA damage response, and probably other cellular pathways, involved in cellular transformation. A large body of evidence indicates that telomerase plays a protective role against damaging agents and stressful conditions and prevents apoptosis (Mondello and Scovassi, 2004; Sung et al., 2005). Although it is still not clear whether telomerase activity is required for this protective role, recently Lee et al. (2008) provided evidence that in mouse cells TERT expression promotes survival and protects from apoptosis independently of its enzymatic activity, possibly blocking the mitochondrial death pathway. Concluding remarks

Telomerase has been discovered as the enzyme deputed to telomere lengthening. Ectopic telomerase expression in somatic cells can allow the bypassing of cellular senescence, leading to cellular immortality. However, several studies indicate that prolongation of cellular lifespan can be associated with activation of cancer-associated pathways; thus, the potential use of telomerase immortalization for therapeutic purposes requires further studies to set up methods suitable to obtain ‘normal’ cells with an extended proliferative potential. Cell lines undergoing neoplastic transformation upon telomerase expression can be useful models for studying molecular changes associated with human carcinogenesis. Response to telomerase activation in normal cells has allowed the identification of telomerase functions, which are independent from telomere lengthening but play a role in tumorigenesis. Multiple roles of telomerase in tumorigenesis have also been highlighted by the phenotypic characteristics of tumor cells in which telomerase activity is abrogated. The many activities of telomerase in tumorigenesis stress the importance of developing therapeutic agents against this complex; in addition, uncovering the pathways regulated by telomerase, and understanding the mechanisms by which this occurs, can provide new targets for cancer treatment. Acknowledgements We are grateful to A. Ivana Scovassi for critical reading of the manuscript.

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