Mechanism of Telomere Induced Senescence

Despite these exceptions to the telomere hypothesis of cell aging and immortalization, the fact that numerous studies have now shown, in many different types of human primary cell cultures, that ectopic expression of Tert is sufficient to restore telomerase activity, prevent telomere shortening, and endow the cells with replica-tive immortality (Harley 2001) clearly shows that telomere shortening is the primary mechanism accounting for the finite replicative life span of normal human cells, at least in vitro. Furthermore, as described above, there is now considerable evidence suggesting that the shortening of just one or perhaps a few telomeres below a critical length, as opposed to the erosion of all telomeres in a cell below an average length, is what triggers cell senescence. The question remains, how does telomere shortening below a critical length signal the induction of replicative senescence ?

To understand how this signal is initiated, it is important to consider why tel-omere shortening ultimately causes replicative senescence. It has been known for some time, from cytogenetic studies by Barbra McClintock in maize, that chromosomal ends in eukaryotic cells are capped by essential genetic elements (i.e., the telomere) (McClintock 1941). We now know that the telomere is a nucleoprotein complex composed of telomeric DNA, the sequence of which is (TTAGGG)n in mammals (Moyzis et al. 1988) and several different telomeric binding proteins (de Lange 2005; see Blasco, this volume). The telomeric binding proteins associate with the telomeric DNA to form a higher-ordered structure that caps the ends of chromosomes and protects the ends from erosion and aberrant end-to-end recombination (de Lange 2005; see Blasco, this volume). At the very end of the telomere there is a 3' overhang of the G-rich (TTAGGG) strand (Chai et al. 2006), which allows the telomere end to loop back on itself and form a structure referred to as a t-loop (Griffith et al. 1999). Thus it is tempting to speculate that telomere shortening signals replicative senescence once there is no longer a sufficient amount of telomeric DNA to form a stable t-loop; however, this has yet to be directly demonstrated. Therefore, other possible signaling mechanisms, including the loss of the 3' overhang, or inability of the telomeric binding proteins to form a stable nucleo-protein complex with a limited amount of telomeric DNA, cannot be ruled out. Nevertheless, there is considerable evidence suggesting that normal somatic cells recognize a critically shortened telomere as damaged DNA, akin to the end of a broken chromosome, for reasons described below.

We have known for over 20 years that the tumor suppressors p53 and the retinoblastoma gene product, Rb, are important for arresting the cell cycle in senescent cells. Early studies showed that DNA tumor viruses that prevent activation of p53 or Rb allow primary human cells to proliferate past the normal Hayflick limit, or M1 checkpoint (Ide et al. 1983, Shay et al. 1991). Furthermore, studies have shown that both p53 and Rb exist in senescent cells in their activated form, phosphorylated (Webley et al. 2000) and nonphosphorylated (Stein et al. 1990), respectively, whereas in mitotically active early passage cells, in S phase, p53 and Rb are present, but in the inactive state. Subsequent studies have shown that in some cells, inhibition of p53 with dominant negative p53 mutants (Bond et al. 1994) or with antisense oligonucleotides that target p53 mRNA (Hara et al. 1991) is sufficient to allow cells to bypass the M1 checkpoint, whereas in other types of cells, inhibition of both p53 and Rb is required (Beausejour et al. 2003). This latter study shows that the genetic pathway for replicative senescence is not exactly the same for all types of human somatic cells.

How does activation of p53 and Rb cause cell cycle arrest? Most senescent primary human cells are blocked at the G1/S phase of the cell cycle, because the cyc-lin-dependent kinases (CDK) that are essential for entry into S phase are inactive (Fukami et al. 1995) or expressed at low levels (Afshari et al. 1993) (Fig. 2.3). A number of different CDK inhibitors (CKI) have now been identified, including p21, p16, p27, and p57. It is now well established that activation of p53, in different types of damaged or stressed cells, including senescent cells, allows the enhanced

Fig. 2.3 The mechanism of telomere-induced replicative senescence. A stable telomere is shown in a t-loop confirmation, in which the end of the chromosome is protected. After a finite number of replication events, the telomere can no longer maintain a stable structure, represented as unfolding of the loop, and consequently the end of the telomere is recognized by DNA damage response sensors. See text for further details (See Color Plate)

Fig. 2.3 The mechanism of telomere-induced replicative senescence. A stable telomere is shown in a t-loop confirmation, in which the end of the chromosome is protected. After a finite number of replication events, the telomere can no longer maintain a stable structure, represented as unfolding of the loop, and consequently the end of the telomere is recognized by DNA damage response sensors. See text for further details (See Color Plate)

transcription of the p21 gene (Herbig and Sedivy 2006) (Fig. 2.3). It has also been shown that in the absence of functional p53, p21 is not induced during culture of HDF (Dulic et al. 2000). Furthermore, an elegant study by Brown et al. has shown that knocking out the p21 gene in HDFs is sufficient to enable these cells to bypass the M1 checkpoint, and allow them to continue to proliferate to crisis (M2 checkpoint) (Brown et al. 1997). Regarding Rb, in most proliferating cells, it is a downstream target of CDKs, which act to phosphorylate and inactivate Rb prior to entry into S phase. The main function of Rb is to sequester the transcription factor E2F, which is responsible for transactivation of a number of genes that are essential for entry into S phase (Fig. 2.3). In senescent cells, Rb is hypophosphorylated (Stein et al. 1990), and therefore E2F activity remains inhibited (Dimri et al. 1994), thereby blocking entry into S phase. Thus in many, but not all, primary human cells, Rb acts downstream of p53 to block entry into S phase in senescent cells.

In addition to p21, another CKI that has been shown to play an important role in maintaining cell cycle arrest in at least some types of senescent cells is p16ink4a (Alcorta et al. 1996). Levels of p16ink4a have been shown to be elevated in senescent human cells (Alcorta et al. 1996). However, in contrast to p21, the kinetics of the appearance of p16ink4a following telomere-induced senescence (rapid; 0-48 hours) is different than that observed when cells are exposed to DNA damage inducing agents (slow; 1-2 weeks) (Sedivy 2006). Therefore, the present view regarding the role of p16ink4a in replicative senescence is that it is activated indirectly, via a DNA damage-independent mechanism, following the critical shortening of telomeres, and functions primarily to help maintain, not induce, the senescent state. The exact mechanism by which p16ink4a is activated in senescent cells remains to be fully elucidated; however, recent research suggests that one mechanism may be via the p38 MAP kinase pathway, allowing the ETS-mediated transcription of the p16ink4a gene (Ohtani et al. 2001), and/or by inhibition of Polycomb group proteins, in particular Bmi1, since repression of Bmi1 by shRNA targeting has been shown to cause upregulation of p16ink4a and cause premature senescence of HDF (Itahana et al. 2003; see Sharpless, this volume). In any event, transcriptional upregulation of p21, mediated by active p53, appears to be the primary means by which cell cycle arrest is initiated by telomere-induced senescence because, as has been shown for some types of primary human cell cultures, p16ink4a is not always present at replicative senescence (Beausejour et al. 2003), knocking down p16ink4a has not been shown to lead to a significantly extended replicative life span (Bond et al. 2004), and some cell strains in which the p16ink4a gene has been mutated still undergo normal replicative senescence (Brookes et al. 2002).

A number of recent studies have also demonstrated that, at the advent of telomere-induced senescence of primary human cells, critically short telomeres are initially sensed by the DNA damage response pathway (Fig. 3.3; see Chang, this volume, and Rudolph, this volume). The DNA damage sensor encoded by the ataxia-telangiectasia mutated (ATM) gene appears to play a particularly important role in initiating the DNA damage response signal, since HDF which lack functional ATM are able to bypass the normal G1/S phase checkpoint at senescence, but then subsequently undergo cell cycle arrest via activation of a G2 phase DNA dam age response pathway mediated by the sensor ATM and Rad3-related (ATR) kinase (Herbig et al. 2004). Other elegant studies have shown, utilizing immunofluorescence and chromatin immunoprecipitation (CHIP) assays, that a number of DNA response factors, including ATM, the histone variant gamma-H2AX, MRE11, the p53 binding protein 53BP1, and the checkpoint kinases chkl and chk2 co-localize with telomeric DNA to form foci in senescent HDF (d'Adda di Fagagna et al. 2003, Herbig et al. 2004). A small number of these foci (1-10) are present in nearly all cells that have undergone telomere-induced replicative senescence, whereas only a very small fraction of early passage cells have detectable foci. These foci are likely directly responsible for activation of p53 and transcriptional activation of the p21 gene in telomere-induced senescence, because p53 is a primary target of these DNA response factors in the presence of damaged DNA (e.g., broken chromosomes). Moreover, targeted inhibition of the chk2 kinase, an important downstream target of ATM, prevents p53 activation and extends the replicative life span of HDF (Gire et al. 2004).

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