Telomeres, telomerase, and myc. An update

Mutation Research 462 Ž2000. 31–47 www.elsevier.comrlocaterreviewsmr Community address: www.elsevier.comrlocatermutres

Telomeres, telomerase, and myc. An update Christa Cerni

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Institute of Tumorbiology-Cancer Research, UniÕersity of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria Received 18 August 1999; received in revised form 3 November 1999; accepted 3 November 1999

Abstract Normal human somatic cells have a finite life span in vivo as well as in vitro and retire into senescence after a predictable time. Cellular senescence is triggered by the activation of two interdependent mechanisms. One induces irreversible cell cycle exit involving activation of two tumorsuppressor genes, p53 and pRb, and the proper time point is indicated by a critical shortening of chromosomal ends due to the end-replication problem of DNA synthesis. The development of a malignant cancer cell is only possible when both mechanisms are circumvented. The majority of human cancers and tumor cell lines produce telomerase, a ribonucleoprotein with two components required for core enzyme activity: telomerase RNA ŽTR. and a telomerase reverse transcriptase protein ŽTERT.. Telomerase adds hexameric DNA repeats ŽTTAGGG. to telomeric ends and thus compensates the progressive loss of telomeric sequences inherent to DNA replication. While TR of telomerase is present in almost all human cells, human TERT ŽhTERT. was found rate limiting for telomerase activity. Ectopic expression of hTERT in otherwise mortal human cells induced efficient elongation of telomeres and permanent cell growth. While hTERT-mediated immortalization seems to have no effect on growth potential and cell cycle check points, it bestows an increased susceptibility to experimental transformation. One oncogene that might activate TERT in the natural context is c-myc. Myc genes are frequently deregulated in human tumors and myc overexpression may cause telomerase reactivation and telomere stabilization which, in turn, would allow permanent proliferation. Is this a general strategy of incipient cancer cells to escape senescence? Several recent observations indicate that other scenarios may be conceived as well. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Telomeres; Telomerase; hTERT; myc; Immortalization; Telomerase in cancer; Alternative lengthening of telomeres ŽALT.

1. Introduction Multicellular organisms have a finite life span that is regulated at the single cell level. Whether this limitation of cell division is a pertinent prerequisite for ageing and death of an organism or rather a cellular safeguard mechanism against uncontrolled proliferation is an open question. ) Tel.: q43-1-4277 ext. 65-245; fax: q43-1-4277-9651; e-mail: [email protected]

Almost four decades ago, Hayflick w1x was the first to report on the in vitro growth of human embryonic cells explanted from various tissues. Initially, cells proliferated for a defined number of generations, but then reduced their mitotic rates and eventually senesced and faded away. A maximal population doubling of approximately 50 was achieved with embryonic fibroblasts, whereas cells from differentiated tissue ceased growing after a few passages. In the meantime inescapable senescence and death as the final fate of normal human cells in

1383-5742r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 Ž 9 9 . 0 0 0 9 1 - 5

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culture has been confirmed by many laboratories and with a variety of cell types and no spontaneous immortalization has ever been observed with human cells. This is at variance with rodent cells where a small fraction of cells is able to escape cell death and develop into established cells lines w2x. Over the last decade, an impressive body of data has been generated to characterize the molecularbiological mechanisms that underlie the initial observations of Hayflick. Genetically determined life span at the single cell level requires two interdependent control systems: one that counts the cell divisions performed and another that irreversibly blocks cell cycle progression. The latter is provided by the action of two main tumorsuppressor proteins, namely p53 and the retinoblastoma protein pRb Žfor review see Refs. w3–5x.. The pathways targeted by p53 and pRb are interconnected by multiple feedbacks and lead to upregulation of p21WA F, a universal cell cycle inhibitor, and dephosphorylation of pRb and pRb related proteins, respectively. The primary goal is the inhibition of D-type cyclins and cyclin D dependent kinases, Cdk4 and Cdk6. Otherwise, activation of these protein complexes initiates phosphorylation of pRb, thereby releasing pRb-bound E2F transcription factor and, as a consequence, transition from G1 to S phase Žfor reviews, see Refs. w6–9x.. Surprisingly, the p53- and pRb-pathways are controlled by a single genetic locus, Ink4arArf w10x, a concept of nature which appears quite risky, as any mutation in this region can simultaneously affect both tumorsuppressor proteins. Ink4arArf encodes two cell cycle inhibitors by using two distinct promoters and alternative reading frames. p16 INK4a prevents phosphorylation of pRb w11x and p19 AR F stabilizes p53 by sequestering the p53-complexing Mdm2 protein into the nucleolus w4,12x. Fig. 1 shows a simplified scheme of the p53- and pRb-pathways controlled by the Ink4arArf locus. Indeed, targeted deletions of the murine Ink4arArf locus relieved cells of both control systems and favoured their immortalization and malignant transformation w10x. Naturally arising mutations of the syntenic region on human chromosome 9p21 have been observed in a variety of human tumors, most notably in melanomas w13x. While knowledge of cell cycle regulation has steadily increased over the last two decades, data on

Fig. 1. Pathways regulated by the Ink4arArf locus. Two different proteins are encoded by the Ink4arArf locus. p16 INK4a inhibits the activation of Cyclin D dependent kinase 4 or 6 ŽCdk4 or Cdk6., which otherwise initiate phosphorylation of pRb. Phosphorylated pRb releases the transcription activation factor E2F, which activates cell cycle relevant genes upon dimerization with other proteins. Murine p19 AR F Žthe human homologue is p14 AR F . sequesters Mdm2 into the nucleolus and thereby prevents Mdm2mediated degradation of p53. The mechanism underlying feedback of p53 on p19 AR F is not known.

the second component of cell senescence, the postulated ‘‘mitotic clock’’, were generated only recently. This was encouraged by the finding that the clock is provided by the ends of chromosomes, the telomeres, and that most cancer cells have reactivated a special enzyme, telomerase, that can reset the clock. The roles of telomeres and telomerase in ageing and cancer have been the subjects of several excellent reviews w14–23x. The present paper will focus on very recent findings in this rapidly developing research field.

2. Structure and function of telomeres Telomeres are special heterochromatin structures that fulfill different functions. They stabilize and protect chromosomes, prevent fusions and recombination, anchor chromosomes within the nucleus and assist the replication of linear DNA Žfor review, see Ref. w24x.. In addition, they also play an important role in chromosome separation during mitosis w25x.

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DNA polymerase replicates DNA only in the 5Xto 3X-direction. Replication is initiated by an 8- to 12-base stretch of RNA hybridized to the template DNA strand. Removal of the terminal RNA primer generates a small, persisting gap at the 5X ends of the newly replicated strands that cannot be repaired. As a consequence, chromosome ends lose at least 8–12 nucleotides for each cell division. In addition, the activity of a putative 5X –3X-exonuclease might remove the terminal 100–200 nucleotides of the C-rich strand of telomeric sequences, leading to long protrusions of single-stranded DNA w26x. A scheme of the DNA end-replication problem is presented in Fig. 2. The 3X overhangs of the very ends of chromosomes are conserved in all higher eukaryotes. A fundamental function of this telomeric region has been found very recently Žsee below.. There is a remarkably conserved sequence organisation of the very ends of chromosomes. This region which exhibits almost no transcriptional activity can be divided into three distinct subregions: Ži. 3X overhangs, Žii. telomeric repeats, and Žiii. telomere associated sequences ŽTAS.. TAS are defined as the areas between the first identifiable single copy gene and the telomeric repeats. Fig. 3 presents the complex array of telomeric regions. The length of TAS varies not only among different species but also

Fig. 2. End-replication problem of DNA synthesis. Conventional DNA polymerase needs a short RNA template Žwhite squares. to start DNA synthesis. The RNA primer is removed upon replication and a stretch of DNA remains unreplicated. Long singleX stranded 3 protrusions are created by the action of a putative X X 5 –3 exonuclease.

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Fig. 3. Organization of eukaryotic telomeres. The ends of telomeres consist of repetitive telomeric sequences with single-stranded X 3 overhangs. Subtelomeric regions are composed of various short satellite-like repeats and middle-repetitive elements, interspersed with telomeric repeats.

between single chromosomes. In human cells, TAS comprise a few up to several hundred kilo base pairs Žkbs. w27x. TAS contain two different types of elements: Ži. relative complex middle-repetitive elements comprising a few to several hundred base pairs and Žii. shorter satellite-like repeats of 20–1000 bp, interspersed in between or within the middle-repetitive complex sequences. There is not only a huge variability with respect to their distribution but also with respect to copy numbers of individual repeats. They rearrange with high frequency and might be responsible for the complexity of single TAS regions. It is assumed that TAS provide a buffer zone between chromosomal very ends and transcriptionally active euchromatin. In contrast to the highly variable composition of TAS regions in various species, the telomeric repeats are remarkably similar. They typically consist of GT-rich repeats, for example ŽTTGGGG. n in the unicellular ciliate Tetrahymena, ŽTTTTGGGG. n in a distantly related ciliate Oxytricha, and ŽTTAGGG. n in mammals. In most species they consist of 6- to 8-bp short monotonously repeated elements. Initially, they were first identified in ciliates due to their great abundance of telomeres. Again, as with TAS, there is a broad variability in the number of repeats ranging from 2 to 3 in ciliates w28x up to several thousands in higher vertebrates w29x. As described for TAS, the amount of telomeric repeats not only varies considerably among species but also between single chromosomes of a given cell. For murine cells, the pure telomeric region comprises up to 60 kb w30x, for human cells the range is between 500 and 2000 5X-TTAGGG-3X repeats. It is currently not possible to accurately measure the length of pure terminal TTAGGG repeats in

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vertebrate cells. Thus, telomere lengths are estimated on the basis of terminal restriction fragments ŽTRF., which encompass the stretch of monotonous telomeric repeats at the terminus and TAS distal to the first restriction enzyme site Že.g., HinfI or RsaI.. TRFs are usually determined either by gel electrophoresis or by chromosomal in situ hybridization. In the former approach, genomic DNA is digested with restriction enzymes that do not cut within the telomeric repetitive sequences. Digested DNA is size-fractionated by electrophoresis and probed with labeled ŽTTAGGG. n oligonucleotides. The variable length of individual telomeres, even of sister chromatids w30x, together with intercellular variations, results in a DNA ‘‘smear’’ on a gel. How do cells discriminate between random DNA breaks and natural chromosome ends? Three mechanisms have been under consideration: Ži. a special protein that caps the 3X telomeric overhangs in order to avoid their misinterpretation of being open DNA strands, Žii. a special structure of the 3X overhangs that knots the very ends of telomeres, and Žiii. special proteins that bind to duplex telomeric DNA and somehow protect the single stranded ends downstream. An example for the first possibility is presented by the alpharbeta heterodimer protein from the ciliate Oxytricha Žfor review, see Ref. w31x., but no mammalian pendant has been identified so far. Also the second theoretical possibility of G–G quadruplex structures of the 3X ends w32x could not be supported by experimental data. Instead, the action of duplex TTAGGG repeat binding proteins appears to solve the problem of protecting open DNA strands in an elegant manner. Two human telomeric repeat factors, TRF1 and TRF2, have been isolated and both proteins appear essential for telomere maintenance. TRF1 binds as a homodimer to duplex TTAGGG and is a negative regulator of telomerase activity w33x. Overexpression of wild type TRF1 causes gradual telomere decline, with a 3- to 11-bp loss per population doubling. Conversely, as shown with a dominant negative mutant of TRF1, loss of functional TRF1 enables telomerase to extend telomeric ends by approximately 35 bp per cell division. The main function of TRF2, which colocalizes with TRF1 during interphase and mitosis, is to maintain the 3X-overhangs at the very end of telomeres w34x. Loss of TRF2 leads to numeri-

X

Fig. 4. Formation of telomere loops. The single-stranded 3 overhang of the very ends of telomeres invades upstream doublestranded telomeric sequences and hybridizes to complementary sequences, thereby forming a displacement loop ŽD-loop.. TRF2 is supposed to stabilize this structure. Telomeric DNA loops out and might be stabilized by TRF1 proteins.

cal and structural chromosomal aberrations, mainly end-to-end fusions after a few cell divisions, and cells senesce or undergo apoptosis w34,35x. The fusions carry telomeric DNA but are devoid of G-rich strands. Analysis of mammalian telomeres by electron microscopy revealed the formation of large DNA loops by hiding the 3X overhang with the aid of TRF1 and TRF2 in the telomeric duplex DNA w36,37x. Fig. 4 shows a schematic presentation of the telomere structure and the presumed action of TRF1 and TRF2. The single stranded TTAGGG overhang folds back and invades upstream telomeric duplex DNA to form a displacement loop ŽD-loop. with complementary AATCCC sequences. This leads to an outlooping of the telomeric double stranded DNA Žt-loop.. While the D-loop at the junction contains only a few hundred nucleotides of single stranded telomeric repeats, the size of the t-loops is large and might encompass the whole telomere. The average t-loop sizes correlate well with the species specificity of telomere lengths, with large t-loops of telomeres separated from murine cells and smaller ones from telomeres of human cells.

3. Telomeres and ageing The telomere hypothesis of aging, first proposed by Olovnikov w38x, postulates that, in the course of multiple cell divisions, the progressive shortening of the chromosomes in a multicellular organism Žsee

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Fig. 2. ultimately signals cell cycle exit. First evidence for this hypothesis was provided by Harley et al. w39x who described the decrease in amount and length of telomeric DNA upon continuous passage of normal human fibroblasts in vitro. Shortening of individual telomeres beyond a critical threshold is incompatible with correct telomere function w39–41x. It is hypothesized that the shortest telomere in a cell rather than the average length may be rate limiting for proliferation. Studies with yeast have shown that the loss of a single telomere is sufficient to induce cell cycle arrest w42x. Senescent cells are characterized by an increased frequency of numerical and structural chromosomal abnormalities, in particular telomeric associations and dicentrics ŽRefs. w34,35,41,43x and references therein.. Human cells from a newborn have TRFs of 7–11 kb, composed of 4–6 kb of true terminal repeats and 3–5 kb of TAS w44x. In cells derived from old individuals the TRFs are significantly shorter. During life span the mean TRF length of human fibroblasts decreases approximately by 1.5 kb representing one third of the telomeric DNA present at birth w44x. When human fibroblasts from individuals of different ages were analyzed for telomere length and in vitro proliferation capacity, a tight correlation was found between these two parameters. The initial age of the donors, however, appeared a less decisive criterion w44x which might be due to different loss rates during infancy w45x. From in vivo and in vitro data it was calculated that the average decrease of TRFs per cell generation is between 50 and 75 bp w19,22,44x. Human somatic cells senesce in a two-stage manner, termed mortality stage 1 ŽM1. and mortality stage 2 ŽM2. w46x. M1 can be overcome by the action of viral oncoproteins such as Simian Virus 40 ŽSV40. large-T w47x or E6 and E7 of ‘‘high risk’’ human papillomaviruses w48x. The M1 senescence barrier is attributed to pRb- andror p53-mediated cell cycle arrest w20,49x. Cells which bypass M1 replicate for additional 20–30 divisions. This growth period is characterized by further loss of telomeric sequences and accumulation of chromosomal aberrations until cells enter M2. Despite the presence of viral oncoproteins, escape from M2 crisis is rare and occurs with a frequency of approximately 10y7 w46x. Analysis of rescued, permanently proliferating cell lines revealed either long or short, but stable telomeres

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Fig. 5. Telomere length in the course of cellular transformation. Telomere lengths decrease with continuous replication of normal cells due to the end-replication problem of DNA synthesis. At a critical telomere length cells enter M1 stage through the action of pRb and p53 and eventually die. Cell death is symbolized by crosses. Upon inactivation of pRb and p53 Že.g., by expression of SV40 large-T., cells continue to divide thereby losing additional telomeric sequences Ž s M2 stage.. A small percentage of cells might survive by reactivation of telomerase, which stabilizes or prolongs telomeres.

w15,23,50x. A schematic presentation of the correlation between telomere length and M1 and M2 stage is shown in Fig. 5. This scenario was seen with many different cell types and at many occasions and supports the hypothesis that escape from limited lifespan is causally linked to the stabilization of chromosomal ends. In contrast to the progressive shortening of the telomere length in mortal human somatic cells, the germ-line lineage is immortal and telomere lengths are maintained throughout life due to highly active telomerase w44x.

4. Function and composition of telomerase As stated above, conventional DNA polymerases cannot fully replicate blunt-ended DNA molecules or eukaryotic chromosomes which contain 3X-overhangs. To prevent continuous loss of telomeric sequences which is essential for the normal function of unicellular organisms or germ cell lineages, an enzyme is required which is capable of de novo synthesis of telomeric DNA. Function and regulation of such an enzyme have been a big mystery until 1985 when Greider and Blackburn w51x succeeded to characterize a telomerase activity in Tetrahymena which

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contained both protein and RNA w52,53x. Telomerase was identified as a ribonucleoprotein with reverse transcriptase ŽRT. activity that carries its own RNA template for the DNA repeats at telomeres. The identification of a very similar biochemical activity in the human cervical cancer cell line HeLa w54x indicated that telomere maintenance by telomerase must be a highly conserved mechanism and might play a role in tumorigenicity.

5. The RNA subunit of telomerase Telomerase activity depends on an essential RNA subunit. The gene for this RNA component has been cloned and characterized from more than 25 species including various ciliates, yeast strains, mouse and human Žfor review, see Ref. w118x.. There is a striking variability in the lengths of all these RNAs. They range from 147 to 191 nucleotides in ciliates w53x to a few hundred nucleotides in mouse and human w55,56x to 1.3 kb in Saccharomyces cereÕisiae w57x. Their common feature is a sequence that is complementary to approximately 1.5 telomeric repeats. Mutations in this region lead to predictable alterations in the newly synthesized telomeric repeat sequences w58x. The templating region of telomerase RNA can be divided into two functionally different parts, one is responsible for alignment with the very last 3–5 telomeric nucleotides and one for the extension procedure to create a new telomeric repeat. Reitinerated translocation of telomerase to the newly created 3Xsequence leads to consecutive rounds of telomere elongation by one repeat each. Fig. 6 illustrates the

action of telomerase. There are additional interactions between telomerase and upstream sites in the telomeric region that determine the processivity of the enzyme w59,60x. Despite the highly conserved function of telomerase, there is a considerable variation with regard to both, the size of the RNA components and their primary sequences among the various species, regardless of their phylogenitical relationship. Human and murine RNA subunits share only a 65% sequence homology w55,56x. The conserved function of the RNA subunit in the light of the obvious lack of similarity in length and primary sequence suggests a stronger conservation of the secondary structure. Although the spatial configuration of telomerase and its RNA component is not known at present, an array of pseudoknot structures, helices and stem-loops of the RNA subunit was postulated that might render the template region accessible for the polymerization reaction Žfor review, see Ref. w18x.. That the different RNA subunits indeed display very similar three-dimensional organization is evidenced from reconstitution experiments. Despite divergence in the primary sequence of the RNA subunits, chimeric telomerases with protein- and RNA-components from different species were found to be active in vivo and in vitro w61–63x.

6. The catalytic subunit of telomerase While the RNA part of telomerase could be fished out of cells by hybridization to telomeric sequences, the search for the protein responsible for RT activity proved more difficult, mainly because the enzyme is

Fig. 6. Elongation of telomere by telomerase. As a first step, three nucleotides of the template region of the RNA component of telomerase X hybridize to the last three nucleotides of the telomeric 3 overhang Ža.. The telomere is elongated by the action of reverse transcriptase of the catalytic component of telomerase Žb.. Reitinerated rounds of hybridization and synthesis lead to successive elongation of the telomere Žc.. Capital letters, ‘‘old’’ telomeric repeat; small letters, newly synthesized repeat.

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in such a scarce supply in most cells. Not before 1996 have two proteins in the ciliate Euplotes aediculatus, p123 and p43, been isolated that copurified with telomerase activity w64x. p123 has been identified as the first representative of the long thoughtafter catalytic subunit of telomerase w65x. The rapid and unequivocal identification was made possible by sequence comparison of p123 with the yeast gene EST 2, which is one of four EST Žever shorter telomeres. genes involved in telomere maintenance. These genes act in the same pathway and mutations in one of them induce a senescent phenotype in yeast w66x. The derived amino acid sequences of p123 and EST2 contain motifs common to all RTs, consistent with the function of telomerase in synthesizing telomeric DNA from an RNA template. Homologues of the telomerase reverse transcriptase ŽTERT. have since been cloned and characterized from several species, including various ciliates, yeast, mouse and human w62,67–71x. Human TERT ŽhTERT., previously referred to as hTRT w68x, hEST2 w69x, and hTCS1 w70x is located on chromosome 5p15.33. Comparison of different catalytic subunits of telomerase with other RTs revealed seven conserved sequence motifs and one domain that is unique for telomerases, termed T-motif w68x. In particular, the three conserved aspartic acids that are involved in nucleotide binding and catalysis in conventional RTs are also essential for telomerase activity w65,67,68x. Overall, the TERT proteins are most similar in structure and function to non-LTR retrotransposons and group II introns and represent a subgroup of RTs w68x. It is notable that telomerase presumably is a large multi-protein complex with the RNA component and the catalytic subunit as the two core components. Other telomerase-associated proteins have been identified such as p80 in ciliates w72x and the human pendant hTPL1 w73,74x, whereas for the ciliate protein p95 w72x a human homologue is still missing. Telomerase activity can be easily determined by the PCR-based TRAP assay Žtelomeric repeat amplification protocol. developed by Kim et al. w75x. A substrate primer to which telomeric repeats can be added is incubated with cell lysate. Elongation of the first primer depends on the amount and processivity of telomerase in the cell lysate. Subsequently, a reverse primer Žs complementary to telomeric re-

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peats. is then added and PCR carried out, resulting in amplification of the telomerase extension products. Amplified products are size-fractionated by gel-electrophoresis. The presence of a 6-base ladder consisting of telomeric repeats is evidence for telomerase activity in a given sample. Apart from a few exceptions Žsee below., most normal human somatic cells have undetectable levels of telomerase activity and do not express TERT w19,39–41,44,49,69,76x or, as with human embryonic tissue, express alternatively spliced, non-functional hTERT transcripts w77x. Interestingly, the RNA component is present in most cells and tissues, albeit in variable amounts w70,77,78x. This indicates that the presence of hTERT protein is the major limiting factor for telomerase activity. Analysis of a series of different tissues and tumors showed that small amounts of hTR are sufficient for telomerase activity, provided that the catalytic subunit is available. Moreover, ectopic expression of hTERT can restore telomerase activity in telomerase-negative cells w62,79,80x.

7. Mice without functional telomerase Although it is evident that telomeres and telomerase are essential regulatory components at the single cell level, a recent publication by Blasco et al. w81x provided evidence that the action of telomerase on constant telomere length appears important for the maintenance of a given species rather than for an individual or its cells. The authors deleted the RNA component of telomerase from the mouse germline ŽmTR y ry . and investigated the fate of several progenies. Unexpectedly, these telomerase-deficient mice were viable and healthy for the six generations analyzed. The average loss of telomere length was at a rate of 4.8 " 2.4 kb per mouse generation, which is similar to that reported for human cells. Calculated on a per cell division basis, this corresponds to 50–100 base pairs for both genera. Only from the fourth telomerase-negative generation onward, increasing numbers of chromosomal abnormalities were observed which is consistent with the assumption that critical shortening of individual telomeres results in structural chromosomal aberrations. Lack of

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telomerase activity did not prevent malignant transformation of telomerase-deficient cells. They could be efficiently immortalized and transformed by viral oncogenes to the same extent as the corresponding wild type cells as evidenced by tumour formation in nude mice. These initial experiments were done with animals that had a p53-wild type background and it was assumed that progressive loss of telomeric sequences might eventually activate the p53-pathway in the most affected tissues such as testis. Indeed, intercrossing mTR y ry and p53 y ry mice delayed the adverse effects of telomere loss for at least two additional generations of mice and enhanced cellular transformation w82x. These data support the concept that shortening of telomere lengths below a critical threshold activates p53-dependent cell cycle arrest and that the lack of functional p53 prolongs the life span of cells while simultaneously increasing the risk for additional mutations. Mice in general have long telomeres Žup to 150 kb. which is sufficient for four to six generations. Nevertheless, normal murine embryonic fibroblasts ŽMEF. in culture undergo a transitory crisis after about 10–15 population doublings. It is obvious that this is not due to critical telomere shortening but rather to upregulation of cell cycle inhibiting proteins encoded by the Ink4arArf locus w10x. This unique barrier can be overcome easily and mutations in p53 or deletions in the Ink4arArf locus are frequently observed in established murine cell lines. Hence, telomerase activity in mice appears important for the maintenance of the species rather than constituting a safeguard mechanism against malignant growth as evidenced by the efficient outgrowth of telomerase-negative tumor cells w81,82x.

8. Telomerase and human cancer Almost all types of human cancer including head and neck tumors, lung cancer, ovarian carcinoma, breast cancer, cervical cancer, gastrointestinal malignancies, renal cell carcinoma, prostate cancer, bladder cancer and others have been analyzed for telomerase activity during the last 4 years w17,75,83– 108x. The vast majority of human tumors have telomerase activity, although little correlation was

found between the presence of telomerase and tumor cell telomere length w75x. The refinement of the original TRAP assay and the recent availability of hTR- and hTERT-specific probes revealed some additional details. It became clear from these studies that a strong association exists between high telomerase activity and tumor progression. This is best documented in clinical situations in which benign, premalignant and malignant pathologies with defined genetic or morphological alterations could be compared, such as colorectal and cervical carcinogenesis. In the case of colorectal carcinogenesis, the percentage of telomerase-positive adenomatous polyps of small, intermediate and large size rose from a few to 20% and to 45%, respectively, and increased to approximately 90% in adenocarcinoma ŽRef. w17x and references therein; w75,97x.. A similar correlation between telomerase activity and malignant progression was found in cervical lesions. The percentage of telomerase-positive cervical swabs increased with the grading of cervical intra-epithelial neoplasias ŽCIN. from 31% for CIN1 to 71% for CIN3 w93,94x. The latter value, in turn, is similar to that found with stages I and II of cervical cancers, whereas almost all tumors of stages III and IV had high telomerase activity w17,94,95x. In a study with proliferative breast lesions, less than 60% of in situ carcinoma were telomerase-positive and the percentage increased to 90% in infiltrating breast carcinoma w91x. These studies suggest that telomerase reactivation is an early event in the malignant development in many tissues and support the hypothesis that telomere maintenance by telomerase reactivation may be a prerequisite for cellular transformation. However, there are notable exceptions. About 20% of advanced carcinomas, irrespective of their origin, lack telomerase activity w15,17,23,75x. Furthermore, Hodgkin lymphoma cells were found invariably telomerase-negative w106x and both, skeletal and soft tissue tumors had only occasionally telomerase activity and only at very late stages w107,108x. The observation of such consistently ‘‘reluctant’’ tumor cell types, together with the fact that at least a fifth of all tumors are telomerase-negative, challenge the hypothesis that efficient tumor cell growth relies on telomerase reactivation. Thus, an alternative telomere lengthening mechanisms ŽALT. was postulated by Bryan et al. w15x which is telomerase-independent Žsee below..

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9. Telomerase induces immortalization If progressive telomere shortening causes cellular senescence of human cells, then ectopic expression of the catalytic component which is rate limiting for telomerase activity should result in an extended life span. Bodnar et al. w80x was the first to report on the efficient reconstitution of telomerase activity by transfer of hTERT into normal human retinal epithelial cells and normal human foreskin fibroblasts. The reconstituted telomerase activity not only resulted in extended telomeres in individual cell clones but, most importantly, was associated with a prolonged life span of the otherwise mortal cells. The efficacy of telomere elongation was dependent on a certain threshold level of telomerase activity that was readily achievable in epithelial cells but only occasionally in fibroblasts. A separate study reported that human mammary epithelial cells were likewise immortalized by ectopically expressed hTERT while the same experimental procedure applied to human fibroblasts yielded senescent cells despite telomerase activity w109x. Thus it appears that in certain primary cell types senescence can only be bypassed by concomitant activation of telomerase and loss of either pRB- andror p53-function w37,49x. It is remarkable that telomerase-rescued human cells, irrespective of their origin, retained all biological features of their normal progenitors w110,111x. Cell cycle regulation, response to various kinds of DNA damages and both, pRB- and p53-dependent cell cycle checkpoints remained unaltered throughout a long period of observation. Thus, it is concluded that with human cells, counterbalancing the loss of telomeric sequences via the action of telomerase confers an indefinite, but normal growth potential. Does this then prone a human cell to experimental transformation? Obviously it is so. A very recent report w112x showed that the combination of hTERT with SV40 large-T and with mutated c-Ha-ras was sufficient to transform human cells of epithelial or fibroblastic origin in vitro. It is worth mentioning that the succession of transfected genomic elements — first SV40 large-T then hTERT then c-Ha-ras — was decisive for eventual cell transformation w112,120x. This is the first evidence that human cells, like rodent cells, can be transformed in vitro with a defined set of oncogenes. Moreover, this tailored procedure revealed that the

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necessary number of deregulated pathways in human cancer cells is lower than previously anticipated. In fact, a controlled step-wise manipulation of human cells is supposed to have an enormous impact on basic and clinical research, with benefits beyond imagination.

10. Telomerase and c-myc In the above-mentioned experiments, cellular immortalization was achieved by either retroviral infection or transfection of plasmidal constructs with strong promoters to drive hTERT expression. Such experimental settings do not reflect the in vivo situation. Thus, the question was obvious: how does telomerase become upregulated in human malignancies? In view of the plethora of genetic alterations — even in less advanced cancers — which affect a variety of distinct signal pathways, the search for relevant genes resembled the search for a needle in a haystack. Therefore, a major breakthrough was the finding that c-myc, a long- and well-known oncogene, activates telomerase w109,113x. The myc gene family encodes transcription factors which, upon dimerization with Max protein, bind to the DNA sequence 5X-CACGTG-3X , termed E-box, or related sequences. Timely controlled c-myc expression is a major prerequisite for G1-S phase transition in most cell types and both canonical and non-canonical E-boxes are found in the regulator regions of many housekeeping genes, including those coding for translational regulatory factors Žfor review, see Refs. w114–117x.. Hence, there is an intimate connection between c-Myc level and DNA metabolism and other metabolic pathways that control cell growth and cell proliferation. Conversely, deregulated c-Myc function by overexpression, amplification, translocation and mutations of myc genes is an obligatory finding in a wide variety of different human tumors Žfor review, see Refs. w114,115,117x.. The contribution of deregulated c-myc to cellular transformation was already recognized 1983 when c-myc was classified as an immortalizing oncogene which cooperated with ras to transform primary embryonic rat cells w118x. However, since then, the genetic mechanisms underlying c-myc-mediated immortalization remained a mystery. Given the importance of proper c-myc regu-

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lation, it was puzzling that up to very recently and despite strenuous efforts no cell cycle relevant gene had been identified whose activation was both cmyc-dependent and directly linked to senescence. Is hTERT a candidate? Possibly. Expression of c-myc in human epithelial and fibroblast cells induces transcription of the endogenous TERT gene leading to telomerase activation and, in turn, to telomere stabilization w109x. Interestingly, the earlier observation that E6 of HPV16 induced pronounced telomerase activity in primary human keratinocytes and mammary epithelial cells w119x could be explained by E6-mediated posttranslational modification of endogenous c-Myc w109x. Genetic analysis of the hTERT locus revealed 29 canonical and non-canonical E-box sequences in the hTERT promoter region and nine in the second intron w113x. This number by far exceeds the frequency of E-box sequences found in other Myc-regulated genes. The E-boxes in the TERT locus are preferred target sites for MycrMax heterodimers und TERT becomes upregulated by overexpressed c-Myc w113,120x. Because activation of TERT by c-myc has been shown to be independent of protein synthesis, TERT is a direct target of c-Myc w120x. The link between myc upregulation and telomerase reactivation appears not to be confined to human cells since transfection of v-myc into telomerasenegative avian cells induced telomerase activity as well w121x. It is worth mentioning that the regulation of the hTERT gene is presumably complex and at present a matter of intense investigation. Multiple signals are likely to act on the TERT promoter and regulate its sustained activity in transformed cells or repress it in normal cells. For example, hybrids between immortal cells that were telomerase-positive and mortal cells that were telomerase-negative had a limited life span. Since these hybrid cells had low telomerase activity, it was concluded that telomerase was significantly inhibited by the normal fusion partner w122x. Putative telomerase repressing genes were found located on human chromosome 3p w123x. Nevertheless, the finding that c-Myc activates hTERT is an important step towards understanding how TERT might be regulated. Are myc and TERT activities interchangeable with regard to immortalization of human cells? Apparently not, although there are only limited data on this

question at the moment. In many cases myc has proven to play an essential role in control of cell growth and differentiation by enhancing or repressing the transcription of genes relevant for the cell cycle Žfor review, see Refs. w114–117x.. Hence, activation of these genes — concomitant with reactivation of telomerase — might be necessary for some cell types to override senescence and crisis. It was observed that the human fibroblast strain IMR-90 could be immortalized by c-myc while hTERT-induced telomerase activation failed to do so w109x. Moreover, TERT was unable to cooperate with ras in a transformation assay with rat embryo cells while c-myc was very efficient in this respect w118,120x. Clearly, more data on this topic will arise in the near future. The implications of the finding that TERT upregulation might be a consequence of myc overexpression are far-reaching. Since ectopically expressed myc can induce telomerase in normal human cells to levels similar to those observed in human tumor cell lines, deregulated myc expression in human malignancies could well account for their telomerase activity. Several data support this concept. A good correlation between Myc levels and telomerase activity was found in neuroblastoma in which amplification of N-myc is a late-stage indicator w124x. In another study on mouse erythroleukemia differentiation, a strikingly similar, coordinated expression pattern of mTERT and c-myc was observed w120x. Conversely, treatment of three different human leukemic cell lines with antisense c-myc oligonucleotides resulted in downregulation of telomerase activity w125x. These data, although limited, demonstrate the close correlation between Myc level and telomerase activity in transformed cells. A dependence of telomerase activity on myc expression can also be inferred from physiological situations, although evidence is only indirect at the moment. A variety of normal, highly proliferating cells or cell compartments Žwith presumably appropriate myc expression. are characterized by significant amounts of telomerase activity. Examples are hematopoietic cells w126,127x, basal cells of the epidermis w128x, the endometrium during the proliferative phase of the menstrual cycle w129x, uterine cervical cells w130x, the oral mucosa w101x and regenerating liver. This indicates that telomerase activity is correlated with

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cell proliferation. Belair et al. w102x studied telomerase activity in isogenic samples of uncultured and cultured specimens of both, normal uroepithelial cells and malignant tissue of the bladder. Uncultured samples met the expectation with regard to telomerase activity, i.e., normal bioptic material was telomerasenegative and samples from tumors were telomerasepositive. However, upon cultivation, telomerase activity was unequivocally linked to the growth of the cells, irrespective of their phenotypes. These remarkable findings were not a peculiarity of urogenital cells as the same results were obtained with specimens of breast and prostate tissue. This suggests that telomerase activity correlates with cell proliferation rather than with the phenotype of a cell. It is possible that the increased myc activity in most tumors could be responsible for the high frequency of telomerasepositive tumors. On the other hand, expression of telomerase does not necessarily lead to elongated telomeres. Broccoli et al. w131x found significant amounts of telomerase activity in normal human blood and bone marrow cells comparable to their malignant counterparts. Yet, these cells have shortened telomeres after in vitro cultivation. In another study, it was found that ectopic expression of hTERT in the normal human fibroblast strain IMR90 averted cell crisis but did not prevent telomere shortening. The resulting immortalized cell line continued to proliferate despite progressive loss of telomeric sequences w132x. These data suggest that human telomerase might have a protective function for telomeres which can be separated from telomere elongation Žsee below..

11. Telomere maintenance without telomerase The most common pathway of normal eukaryotic cells to maintain telomere lengths, as exemplified by germ cell lineages or unicellular organisms, is activation of telomerase. Yet, this is not a universal strategy. Indeed, there exists a variety of examples from various species where loss of chromosomal end sequences caused by replication is balanced by other mechanisms. Although yeast cells maintain their telomeres mainly by the action of telomerase, recombinational pathways can be used as well for the same

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purpose under certain circumstances. In S. cereÕisiae, mutations in genes that contribute to telomerase activity resulted in gradual loss of telomeric sequences followed by chromosomal loss and eventual death. However, a small subpopulation of mutant cells survived and sequence analysis revealed the presence of subtelomeric repeats added to almost all chromosomes w133x. Telomere elongation by recombination of TAS sequences is also used by fission yeast when the catalytic subunit of telomerase is mutated w134x. This indicates that recombination over quite long distances in the DNA can substitute for telomerase activity. As already mentioned above, the percentage of human tumors that do not express telomerase activity is on average about 20% w15,17,75,106,107x. For in vitro established human cell lines this percentage is even 40% w135x. Despite the lack of detectable telomerase activity, some of these cell lines analyzed had quite long telomeres and were even more heterogeneous in their telomere lengths than telomerasepositive ones. The TRF pattern of these lines did not change upon extensive passaging which indicates that telomeres were maintained. The mechanism for the constant lengthening is unclear but might require recombinational mechanisms. Some other telomerase-negative cell lines obviously use a different strategy to prevent a critical shortening of their telomeres. Studies on the fate and dynamics of a single marked telomere in a telomerase-negative human fibroblast cell line revealed a burst of telomere elongation alternating with telomeric shortening w136x. A similar dynamic of telomere lengths was reported for two immortalized human cell lines in which telomerase activity had been inhibited with various RT inhibitors w137x. Irrespective of the presence and chemistry of the drugs, telomere lengths as determined at regular intervals increased and decreased in an unpredictable manner. Such discontinuous elongation by non-reciprocal recombination of terminal and subterminal sequences was described for lower dipterans w138x. The fruit fly Drosophila melanogaster lacks canonical telomeric repeats but possesses two retrotransposible elements, HeT-A and TART, which are added not only to chromosome tips but also to broken chromosome ends without specific sequences at the target sites. Surprisingly, little or no cross reactivity was found in other drosophilid flies,

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neither with TTAGGG nor with HeT-A and TART sequences. It was hypothesized that other Drosophila species use different yet unidentified classes of transposable elements which guarantee telomere maintenance either upon continuous or occasional recombination. It must be emphasized that telomere elongation by both telomerase and retrotransposition requires reverse transcription. One might speculate that telomerase with its RT activity is the common ancestor and that the use of DNA recombination represents an alternative bypass mechanism, which is applied when telomerase has been lost as in the case of dipterans. Human cells, however, might use either of them.

12. Myc and ALT There is no doubt that in normal somatic cells telomeric sequences are lost upon repeated DNA synthesis and that shortening of individual telomeres beyond a threshold length signals cell cycle exit and triggers cellular senescence. This is definitely a major safeguard mechanism against uncontrolled proliferation of human cells. A potential human tumor cell surmounts this critical situation by reactivation of telomerase. It appears that there are two distinct modes of how telomerase can contribute to permanent cell proliferation, one that relies on its constant presence and one that requires a transient burst of activity. In the former, a constantly high telomerase activity elongates TRFs to a certain extent or provides at least stabilization of short telomeric ends. In these cases where telomere maintenance is exclusively dependent on this enzyme activity, long-term inhibition of telomerase will sooner or later lead to a senescent phenotype. There is only one report where this was investigated systematically in human cells. When HeLa cells are treated with oligonucleotides tailored to inhibit the RNA component of telomerase, some clones died after 23–26 population doublings w56x. It appears pertinent to apply similar experimental procedures to other tumor cell types in order to appraise the potency andror limitation of future telomerase inhibitors as therapeutic agents. It also remains to be determined whether permanent presence of telomerase activity is dependent on constitutive myc expression andror due to deregulation of

other telomere-associated proteins, e.g., TRF1, which modulate telomerase activity. The second possibility to protect short telomeres during senescence, requires a modest and perhaps transient reactivation of telomerase. It was shown very recently that ectopic expression of hTERT was able to avert cell crisis and extended the lifespan of human cells, yet their telomeres continued to shorten w132x. It was suggested that in this case telomerase provided a kind of capping function for telomeres. Although details of this telomere protection by telomerase are not known at the moment, it might turn out to be a more essential function of telomerase than initially anticipated. As a first support of the hypothesis that transient activation of telomerase might be sufficient to overcome cell crisis under certain circumstances, it was recently reported that one clone of SV40-immortalized human fibroblasts had low telomerase activity during cell crisis, but was found telomerase-negative after a few passages w139x. It is speculated that telomerase-negative tumors might also have experienced such short periods with a burst of telomerase activity at some stage during their development which remained undetected due to its transitory nature. In view of the recent report that normal human cells can be intentionally transformed by a defined combination of genes, comprising hTERT and two oncogenes w112x, the inclusion of inducible TERT constructs in similar experiments would be of great value. It stands to reason that the fate of potential cancer cells with only a burst of telomerase activity during crisis will depend on the cell’s success to activate ALT mechanisms. The proper timing of activation of either non-reciprocal or reciprocal recombinational mechanisms will be a prerequisite for permeant growth. The feasibility to activate ALT obviously depends on the cell type, as mesenchymal tumors use ALT mechanisms much more frequently than cancers originating from epithelial cells w135x. A contribution of myc in this context appears redundant although direct evidence is lacking. However, it seems unlikely that on one hand myc is activating TERT during crisis which is characterized by reduced cell proliferation and on the other hand relieves TERT from upregulation in the post-crisis phase when cells eventually resume growth. It is conceivable that deregulated myc — as seen in

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many different tumors — contributes to enhanced proliferation thereafter and one consequence is a concomitant, but unessential upregulation of TERT. At first, the finding that TERT is a downstream target of c-Myc appeared a major step towards understanding the role of telomerase activity in the process of malignant transformation. In fact, it raises more questions than it provides answers because the apparent superposition of telomerase activity on telomerase-independent telomere lengthening mechanisms is providing an unexpected additional level of complexity. Nevertheless, it represents an exciting topic for future research. Acknowledgements I thank Siegfried Knasmuller ¨ and Christian Seelos for reviewing the manuscript and Soleman Sasgary and Monika Breit for expert preparation of the figures. This work was supported by the Herzfelder’sche Familienstiftung. References w1x L. Hayflick, P.S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res. 25 Ž1961. 585–621. w2x G.J. Todaro, H. Green, Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines, J. Cell Biol. 17 Ž1963. 299–313. w3x G.W. Kaelin Jr., The emerging p53 gene family, J. Natl. Cancer Inst. 7 Ž1999. 594–598. w4x D.A. Freedman, A.J. Levine, Regulation of the p53 protein by MDM2 oncoprotein — 38th GHA Clowes Memorial Award Lecture, Cancer Res. 59 Ž1999. 1–7. w5x X. Grana, J. Garriga, X. Mayol, Role of the retinoblastoma protein family, pRB, p107 and p130 in the negative control of cell growth, Oncogene 24 Ž1998. 3365–3383. w6x R. Parwaresch, P. Rudolph, The cell cycle — theory and application to cancer, Onkologie 19 Ž1996. 464–472. w7x M.D. Planas-Silva, R.A. Weinberg, The restriction point and control of cell proliferation, Curr. Opin. Cell Biol. 9 Ž1997. 768–772. w8x N. Dyson, The regulation of E2F by the pRB-family proteins, Genes Dev. 12 Ž1998. 2245–2262. w9x D.G. Johnsons, C.L. Walker, Cyclins and cell cycle checkpoints, Annu. Rev. Pharmacol. Toxicol. 39 Ž1999. 295–312. w10x T. Kamijo, F. Zindy, M.F. Roussel, D.E. Quelle, J.R. Dowing, R.A. Ashmun, G. Grosveld, C.J. Sherr, Tumorsuppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF, Cell 91 Ž1997. 649– 659.

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