Key words: telomere, telomerase, senescence, Hayflick limit, fibroblast, human

Summary
Telomeres are well established as a major "replicometer", counting the population doublings in primary human cell cultures and ultimately triggering replicative senescence. However, neither is the pace of this biological clock inert, nor is there a fixed threshold telomere length acting as the universal trigger of replicative senescence. The available data suggest that opening of the telomeric loop and unscheduled exposure of the single-stranded G-rich telomeric overhang might act like a semaphore to signal senescent cell cycle arrest. Short telomere length, telomeric single-strand breaks, low levels of loop-stabilising proteins, or other factors may trigger this opening of the loop. Thus, both telomere shortening and the ultimate signalling into senescence are able to integrate different environmental and genetic factors, especially oxidative stress-mediated damage, which might otherwise become a thread to genomic stability.

1. Introduction: replicative senescence in primary human cell culture
Replicative senescence as first described for human fibroblasts [1] is the permanent loss of replicative potential, which is rather independent on culture time (however, see [2]) but occurs after a more or less constant number of cell divisions ("Hayflick limit") under constant culture conditions. In contrast to quiescence, the senescent cell arrest is stable in human cells. On the other hand, senescent cells stay alive for many months and are not preferentially sensitive to inducers of cell death. Manipulations that render p53/p21 and p16/p19/pRb pathways non-functional can overcome senescence (for review see [3,4]), but will result only in a limited elongation of life span in human cells (characterised by further telomere shortening) before a second „mortality stage" or crisis [5] occurs.

The stability and reproducibility of the Hayflick limit has led to the concept that replicative senescence in human cells is governed by an intrinsic cell division counting mechanism, a "replicometer". This is in contrast to the situation in primary rodent cell culture, where the facts that there is no correlation between telomere length and senescence-like arrest, and that culture conditions can be found which allow apparently unlimited growth of certain primary rodent cell cultures without compromises in check point functions [6,7] led to the suggestion that the senescence-like arrest in rodent cells is due to extrinsic stresses ("culture shock", [4]). While this discussion is outside the scope of the present review, it should at least be mentioned that both the Hayflick limit and the rate of telomere shortening are not independent from extrinsic stresses in human cells as well [8-14].

2. Telomeres trigger replicative senescence
There are three principal, widely confirmed observations to support the hypothesis that telomeres are the replicometer, which counts cell divisions and ultimately triggers replicative senescence: First, telomeres shorten with each population doubling in primary human cell cultures but stop shortening in non-dividing cells [15-17]. Second, immortal cells, being it single cell organisms, germ line cells or tumour cells express in their vast majority active telomerase, the enzyme that binds to the single-stranded 3' end of the telomere and re-elongates it [18]. Immortal cells without telomerase activity maintain long telomeres by a recombination-based mechanism [19]. Third, human fibroblasts, which display telomere shortening and senescence, can be immortalised solely by transfection with the catalytic subunit of human telomerase, hTERT. This transfection results in the restoration of functional telomerase from exogenous hTERT and the endogenous template RNA, hTR [20], elongation of telomeres and growth far beyond the Hayflick limit of these strains [21,22]. Even after prolonged culture, these strains were normal in checkpoint control and did not show signs of a transformed phenotype [23,24]. There are evidently other potential replicometer besides telomeres, for instance the progress

ive decline in DNA methylation in senescing cultures, which is able to activate p21 and to induce a senescence-like arrest [25]. In human epithelial cells, the pRb pathway is an additional block towards immortalisation, even if telomere shortening is compensated for by hTERT transfection [26]. However, the present results clearly identify telomeres as a major determinant of replicative senescence in human cells.

3. How does telomere length trigger replicative senescence?
Still, the signalling pathway(s) from shortened telomeres to induction of replicative senescence are largely unknown. In yeast, silencing trans-acting factors like SIR3 and SIR4 are part of telomeric heterochromatin and repress telomere-adjacent genes as they spread along the chromosome [27]. In theory, de-repression of these genes due to telomere shortening could activate cell cycle blockers. However, experimental evidence for this mechanism to contribute to replicative senescence in human cells is scarce.

On the other hand, the well-demonstrated dependency of replicative senescence on the p53/p21- (and p16/p19/pRb-) pathway strongly suggests that telomere shortening at a certain threshold is recognised as a DNA damage signal. Conceptualising on the observation that telomeric fusions, i.e. dicentric and ring chromosomes, are seen at a relatively high frequency at senescence, Vaziri & Benchimol [28] suggested chromosome breaks during the next mitosis after dicentric or ring formation as the type of DNA damage that triggers senescence. However, it is not clear whether such rather coarse chromosomal events are really necessary as triggers.

Recent data on telomeric higher-order structure suggests a more satisfying mechanism (see Fig. 1): The very end of the telomere is a single-stranded G-rich 3´overhang [29], which is normally protected by intercalation into the double-stranded telomere DNA, forming a telomeric loop [30]. This loop is stabilised by certain telomere-binding proteins, notably TRF1, TRF2 and TIN-2 [30, 31]. It was suggested that unscheduled opening of this loop, exposing the single-stranded telomeric overhang to DNA damage sensors in the nucleus, might trigger senescence [30].

Inhibition of TRF2, which probably contributed to the opening of the telomeric loop, resulted in the activation of a DNA damage checkpoint, which was ATM- and p53-dependent [32]. In parallel, we had shown that transfection of human cells with short G-rich telomeric oligonucleotides, but not with telomere-related or unrelated C-rich single-stranded DNA, triggered a p53-dependent arrest [33]. One would expect that telomere shortening could gradually decrease the probability of formation of a telomeric loop and increase the probability of its unscheduled opening. Depending, among others, on the expression levels of the stabilising proteins, there might be a certain threshold length at which the stability of the loop would become seriously inhibited. The fact that the expression level of TRF2 increases with fibroblast age [34] might indicate a greater demand for this protein to maintain loop stability.

So, the telomere signalling function might be compared to an old-fashioned semaphore: As long as the arm (the single-stranded G-rich overhang) is protected from view, no signal is given and the cell cycle may proceed. If, however, the arm is exposed, braking is signalled. The important feature of this concept is that telomere shortening is one but not the only means to set the signal.

4. Does a threshold telomere length trigger replicative senescence?
It became almost a dogma in telomere research that a certain threshold telomere length is the trigger for replicative senescence. However, what does that mean? Is this threshold the same in different human cells or at least in different human fibroblast strains? Does it apply to the average length of all telomeres, to the length of the shortest telomere, to the length of a marker telomere (whatever this might be) or to the average length of a group of telomeres (e.g. the shortest)? Unfortunately, there are experimental data in favour of, as well as in contradiction to, each of these possibilities.

The average telomere length has been shown to be a good predictor of replicative capacity [16, 35], and its variation between a number of fibroblast strains was smallest at senescence [17]. Moreover, senescence in WI-38 fibroblast under basal culture conditions occurred at the same average telomere length as premature senescence under mild chronic oxidative stress [11]. On the other hand, the average telomere length was clearly different between some other fibroblast strains at senescence [34, and von Zglinicki, unpublished] and MRC-5 fibroblasts treated with an oxygen radical scavenger senesced at higher PD but with longer telomeres than untreated controls [12]. In fibroblasts immortalised by limiting amounts of telomerase, it is not the average telomere length, but the length of the shortest telomeres that is maintained [36]. However, short telomeres accumulate on some chromosomes many PD before senescence. While the longer telomeres still shortened with each population doubling, no further attrition of the short telomeres could be seen and a signalling telomere could not be identified [35].

With respect to telomere signalling, there is also very interesting data from telomerase-expressing tumour cells. It was shown that expression of telomerase extends the lifespan of virus-transformed human fibroblasts without net telomere lengthening [37]. Inhibition of telomerase in tumour cells can lead to an immediate signalling into an apoptosis pathway (senescence being most probably compromised in tumour cells) without prior telomere shortening [38, Saretzki et al., in prep.]. These data together with similar results from yeast support the idea that one way of preventing a senescence signal to emanate from telomeres is the capping of their ends by active telomerase [39]. Together, these data make very clear that the idea of a fixed threshold telomere length as the universal and unique trigger of replicative senescence is far too simple. However, the "semaphore concept" might be able to integrate the different facts. For instance, capping of telomeres, i.e. the binding of telomerase to the G-rich overhang would appear as a second line of defence in those cells where due to telomere shortening or any other means the telomeric loop would be unstable and the probability of "semaphoring" would be high (Fig. 1C). Furthermore, telomere length would be a major determinant of whether a cell cycle arrest signal originates from the telomere or not, but not the only one. Variations in absolute and relative contents of telomere-binding proteins would be another one. Also, one would expect that torsional stability of the telomeric loop would be important. That means that telomere-specific actions of topoisomerases as well as the induction of single-strand breaks in the telomeric loop could contribute to signalling [40]. A specific interaction of topoisomerases with telomeres has been reported [41] as well as the induction of a senescence-like arrest by topoisomerase poisons [42]. Telomerase-expressing cells display a decreased sensitivity towards topoisomerase poisons as compared to their isogenic counterparts [38]. Telomeres are less efficient in single-strand break repair than the bulk of the genomic DNA [43]. The resulting accumulation of telomeric single-strand breaks before DNA replication causes a large fraction of the telomere shortening observed in many human fibroblast strains [2, 12, 13]. Telomeric accumulation of single-strand breaks has been observed in quiescent and senescent cells [2, 11]. Whether there is a threshold value of telomeric damage that induces senescence without prior telomere shortening has not been established yet.

Conclusions
Telomeres are well established as the major replicometer, counting the number of cell divisions and eventually signalling replicative senescence in primary human cell strains. However, this does not mean that the pace of this biological clock is constant. Rather, telomere shortening is largely dependent on the interplay of oxidative stress/antioxidant defence [12, 13, 40]. Moreover, there is no inert threshold length at which senescence is triggered. Rather, the telomeric loop seems able to integrate different factors, e.g. length, DNA strand breaks, or protein levels, into a common signalling pathway triggered by the opening of the loop. Instead of being simple counting devices, telomeres appear to be highly efficient sentinels to protect cells and tissues from the consequences of potentially genotoxic insults.

Acknowledgements
Work from my group of relevance for this review has been supported by grants from the Deutsche Forschungsgemeinschaft, the VERUM Foundation for Behaviour and Environment, and the Newcastle Hospital Special Trustees Fund.

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Fig 1. A hypothetical model of telomeric structure and signalling function. A) Telomeres form a so-called t- (or telomeric) loop by intercalation of the single-stranded 3'-overhang into the telomeric double helix (forming a D- or displacement loop). The t-loop is stabilised by certain proteins, notably TRF-1, TRF-2 and TIN-2 (modified from [30]). B) Telomere shortening, damage to or loss of telomere-binding proteins or loss of torsional stability due to telomere DNA damage might lead to unscheduled opening of the loop. This exposes the 3' overhang to DNA damage recognition protein(s) (indicated by the question mark). This stabilises p53 and activates a cell cycle checkpoint response. C) Opening of the loop will not trigger cell cycle arrest if the single-stranded 3'-overhang is protected by telomerase (TEL) capping [39]. For additional details, see text.

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