The p53/p21 pathway has clear role during induction of senescence. Mouse embryonic fibroblasts (MEF) lacking either p53 (Harvey and Levine, 1991) or p19 (Kamijo et al, 1997, 1999) do not senesce. Human cells bypass senescence when both the p53 and the Rb pathway is inhibited by e.g. Simian virus 40 (SV40) large T antigen (Stein et al, 1990). Furthermore, senescence induced by inactivation of SV40 large T antigen can be inhibited by introduction of a dominant negative p53 in some cell types (Fujii et al, 1999). Interestingly, inhibition of the p53 pathway in cells already senescent can reverse the phenotype as injection of anti-p53 antibodies (Gire and Wynford-Thomas, 1998) or a SV40 large T antigen that only binds and inactivates p53 (Beausejour et al, 2003), can reinitiate DNA synthesis at least in some cell types. Also, overexpression of p53 can induce senescence in some tumor cell lines (Wang et al, 1998). The downstream target of p53, p21 can induce senescence in tumor cell lines independent of p53 status (Fang et al, 1999; Wang et al, 1999; Chang et al, 2000a), and human but not mouse fibroblasts lacking p21 bypass senescence (Brown et al, 1997; Pantoja and Serrano, 1999; Wei et al, 2001). This indicates an important role of p21 for induction of senescence in human cells and further indicates that induction of senescence differs between species. Reactive oxygen species (ROS) are possible mediators of the senescence response downstream of p53/p21 (Polyak et al, 1997). In support for an involvement of ROS, both p53- and p21-induced senescence has been shown to be at least partly dependent on ROS (Macip et al, 2002, 2003).

The telomere is a structure located at the end of each chromosome. It consists of a repeated DNA sequence (TTAGGG.) and associates with several binding proteins. There are species variations in telomere biology, for example humans have shorter telomeres compared to mice (Kipling and Cooke, 1990; Wright and Shay, 2000). Telomerase, a reverse transcriptase, can extend the telomere DNA by using a nucleus-encoded RNA as a template for its RNA-dependent DNA polymerization (Blackburn, 1992). Each telomere ends with a 3¢ single stranded sequence of about 200 nucleotides that folds back to the double stranded telomere sequence to form a loop structure called the t-loop (Griffith et al, 1999; Wright et al, 1997). The t-loop together with the telomere binding proteins are believed to protect and hide the end of the telomere to avoid a DNA damage signal and/or regulate the length of the telomere.

Telomere-induced senescence is thought to result from the “end-replication problem”. The replication machinery can not start at the absolute end of the chromosome and therefore a piece of the telomere is lost following each round of replication (Levy et al, 1992). Recently, t-loop sized deletions of the telomere have been detected in primary cells indicating that other mechanisms could contribute to telomere shortening (Wang et al, 2004a). The rate of telomere shortening is suggested to be related to the length of the single stranded telomere DNA. However, this may differ between cell lines, where telomere shortening can be proportional to the length of the single stranded DNA, and primary cells from different donors, where no correlation between lengths of the single stranded DNA and telomere shortening was found (Huffman et al, 2000; Keys et al, 2004).

The main experimental evidence that the telomere drives replicative senescence reflects experiments where overexpression of telomerase in several cell types allows those cells to bypass senescence (Bodnar et al, 1998; Vaziri and Benchimol, 1998; Yang et al, 1999; Zhu et al, 1999; Ouellette et al, 2000b; Steinert et al, 2000; Rufer et al, 2001; Wood et al, 2001; Harada et al, 2003). However, in some cell types telomerase fails to overcome senescence as these cells senesce for other reasons then telomere shortening (see below) (Kiyono et al, 1998; Dickson et al, 2000; Farwell et al, 2000). Initially it was uncertain what overexpression of telomerase really achieved as cells with telomerase (that escaped senescence) showed shorter telomeres than senescent cells (Zhu et al, 1999; Ouellette et al, 2000a). A reasonable explanation for this paradox was that it is not the average telomere length that determines when a cell enters senescence, rather senescence is triggered by the shortest telomere within each cell (Hemann et al, 2001; Baird et al, 2003; der-Sarkissian et al, 2004). Interestingly, telomerase was initially reported to have no other role beyond maintaining the telomere (Jiang et al, 1999; Morales et al, 1999). However, it is now clear that this is not true as telomerase can contribute to tumorigenesis e.g. by mechanisms unrelated to telomere elongation (Stewart et al, 2002), stimulate proliferation (Smith et al, 2003) and change the response to TGF-b (Stampfer et al, 2001). It seems plausible that these other activities of telomerase could be important for the escape from senescence or the role telomerase has in tumor development (90% of all tumors overexpress telomerase, see Senescence and cancer).

A critical question is what happens to the telomere when it becomes shorter and shorter. Currently there are two main theories, and although they do not seem to rule each other out, it has led to a lot of controversy within the field. The first theory postulates that, upon shortening of the telomere, the proteins that usually cap the telomere are no longer able to protect and a DNA damage response is initiated. Inherited with the model is the assumption that the proteins that bind to the telomere suddenly are unable to do it, presumably because the telomere is too short. The second theory states that the single stranded telomere DNA is degraded or eroded; and that this makes that telomeric structure unstable and induces senescence. The telomere erosion theory does not imply that the telomere has to be particularly short, just that the single stranded overhang is lost. The erosion theory was initially supported by the finding that the single stranded DNA is lost during senescence (Stewart et al, 2003). However, recently this finding has been challenged, as no loss of the single stranded DNA was found in senescent cells (Chai et al, 2005). This clearly questions the basis for the erosion theory. The difference between the two studies did not reflect the selected cell types, but may be related to the different methods used to measure single stranded telomere DNA lengths. The main evidence supporting that senescence is induced when the telomere proteins fail to protect the telomere structure, comes from experiments with TRF2 which binds to the telomere. Overexpression of TRF2 leads to shortening of the telomere but a delayed senescence which indicates that excess TRF2 can maintain a proper telomere structure of short telomeres and that this is a key event that regulates onset of senescence (Karlseder et al, 2002). Also, experiments with a dominant negative TRF2 lead to induction of senescence without loss of telomere DNA (Stansel et al, 2001).

In the last two years significant progress has been made through identification of the signaling pathways that are activated by the telomere and induce senescence. Several groups have identified a DNA damage “response” specifically originating from the telomere structure in senescent cells (Bakkenist et al, 2004; d'Adda di Fagagna et al, 2003; Takai et al, 2003). All studies detected e.g. a phosphorylated form of histone H2AX and several DNA damage related proteins. It therefore seems likely that the telomere structure is identified as a double strand break that signals through ATM, which can phosphorylate H2AX as well as CHK2 (Gire et al, 2004). If telomere senescence is initiated through the ATM pathway, one might ask what maintains the senescent state. Previously it has been shown that it is possible to reverse the “irreversible” growth arrest, at least in some cell types, by inhibiting p53 (Beausejour et al, 2003; Gire and Wynford-Thomas, 1998), and it may therefore be plausible that the DNA damage signals from the telomere still persists in the fully senescent cell (d'Adda di Fagagna et al, 2003). However in another cell type, the DNA damage signals disappeared once the cell became fully senescent (Bakkenist et al, 2004). The cells that maintained an active DNA damage response also demonstrate reversal of senescence by inhibition of p53 (Beausejour et al, 2003). The cells that did not maintain the DNA damage signals have not yet been assessed for the irreversibility phenotype, but it seems possible that there might be cell type differences in this characteristic. Another group has challenged whether the DNA damage signals originate from the telomere and claim that they are randomly distributed upon induction of senescence (Sedelnikova et al, 2004). However the data presented by d'Adda di Fagagna et al. included several methods to assess where the signals originate from (d'Adda di Fagagna et al, 2003).

Based on current data, a likely model for senescence downstream of the telomere could be as follows: (i) the telomere structure and/or function is compromised as a result of telomere shortening, (ii) the DNA ends of the chromosome become exposed and trigger a DNA damage response through the ATM pathway, (iii) depending on the cell type this DNA damage is un-repairable and the ATM signalling persists and maintains the cell in a non-dividing state or the DNA damage is repaired but other pathways have been activated, and the cell maintains an irreversible growth arrest.

What is downstream p53/p21 activation? Several microarray studies have looked at the transcriptome induced by p53/p21 or used bioinformatics approaches to identify p53 responsive genes (Giaccia and Kastan, 1998; Chang et al, 2000b; Zhao et al, 2000; Hoh et al, 2002; Wells et al, 2003; Zhang et al, 2003, 2004; Larsson et al, 2004). New method-developments have enabled large functional screens for genes essential for senescence using siRNAs. Using a reversed genetics approach in the temperature sensitive Simian virus 40 large T antigen model, five genes were identified as being essential for senescence (Berns et al, 2004). Although the identity of several of them was surprising, it indicates that senescence relies on large changes in basic cellular mechanisms. Another protein (Smurf 2) has been identified in a microarray study of replicative senescence, and seems to be specifically induced during telomere senescence compared to hydrogen peroxide induced senescence. Smurf2 can induce a senescent arrest through either the p53/p21 or the p16/Rb pathway but seems unlikely to be essential (Zhang and Cohen, 2004).

V. Oncogene-induced senescence

Activation of oncogenes renders the cell self sufficient in growth signaling and is important for cancer progression (Hanahan and Weinberg, 2000). Interestingly, overexpression of several oncogenes also induce senescence in vitro and this may be an important strategy to avoid cancer progression. RAS (Serrano et al, 1997) and its downstream targets RAF (Lin et al, 1998) and MEK (Zhu et al, 1998), as well as ERBB2 (Trost et al, 2005), eIF4e (Ruggero et al, 2004) and E2F in some cellular contexts (Dimri et al, 2000; Lomazzi et al, 2002) can all induce senescence. This indicates that senescence could be a general defence against activated oncogenes. RAS-induced senescence is best described in the literature but is species specific.

RAS-induced senescence in MEFs is dependent on both p16/RB and p53 (Serrano et al, 1997) and p53 activity alone is not sufficient to induce a full senescent phenotype in MEFs (Ferbeyre et al, 2002). Further, in MEFs with functional p53, disruption of both Rb and the Rb-family member p107 was necessary to avoid senescence but not sufficient to induce transformation (Peeper et al, 2001). The activation of p53 and induction of senescence downstream of RAS in MEFs is dependent on Dmp1, which activates p19 through a Ets site in the INK4A promotor (Sreeramaneni et al, 2005). Therefore, it seems like both a functional p53 and Rb pathway is necessary to induce senescence in MEFs when RAS is overexpressed.

In human cells, RAS does not seem to be dependent on p53 to induce senescence as it was either unchanged (Benanti and Galloway, 2004; Takaoka et al, 2004) or was upregulated but not essential for RAS-induced senescence (Serrano et al, 1997; Wei et al, 2001). Some of the discrepancies of p53 induction during RAS-induced senescence may be related to the dual action of RAS on p53 status as RAS activates both MDM2 and p19 which inhibits and activates p53 respectively (Ries et al, 2000). The outcome of RAS on p53 status may therefore be cell type and experimental condition specific. In support for a non essential role of p53 is RAS-induced senescence, fibroblasts lacking p21 entered senescence following RAS overexpression similarly to p53 negative cells (Wei et al, 2001).

Unexpectedly the stress activated kinase p38 seems to have an important role in human RAS-induced senescence. p38 is activated as a consequence of RAS expression and inhibition of p38 by a small molecule inhibitor (for isoforms a and b) bypassed RAS-induced senescence (Wang et al, 2002). There is data indicating that the activation of p38 could be related to an accumulation of reactive oxygen species as human fibroblasts overexpressing RAS did not senesce at low oxygen levels or in the presence of scavengers (Lee et al, 1999). It is also possible that overexpression of RAS changes the metabolic balance in the cells which activates p38. The later suggestion comes from a study where overexpression of an enzyme needed for glycolysis bypassed RAS-induced senescence in MEFs (Kondoh et al, 2005). The contribution of cell stress to RAS-induced senescence is further supported by a recent report where cells that showed lower stress levels (measured by p16 activity) before addition of RAS, did not senesce after RAS overexpression (Benanti and Galloway, 2004). Together these data favor a model in human cells where overexpression of RAS leads to an accumulation of ROS and increased p38 activity which could activate the p16 pathway; and induction of senescence. It is also possible that both p16 and p38 are activated by ROS in a parallel pathway and that the activity of both is necessary to induce senescence. In support of this model, fibroblasts from a melanoma prone family with p16 deficiency, but functional p19, did not senesce when RAS was overexpressed (Brookes et al, 2002); and fibroblasts from a patient with bialelic mutations of the INK4a/ARF locus showed resistance towards RAS-induced senescence (Huot et al, 2002).

What then is the link between RAS/stress and activation of p16? Several transcription factors has been suggested to be mediators of RAS-induced senescence as they regulate transcription of p16, either positively or negatively. The first class represses transcription of p16 and would be expected to delay senescence when senescence is mediated by p16. Accordingly, the Id proteins have been shown to delay senescence in cell types where senescence is mainly p16 dependent (Alani et al, 1999; Nickoloff et al, 2000; Tang et al, 2002). Similar functions have been established for BMI-1 which inhibits both senescence and apoptosis induced by p19 (Jacobs et al, 1999a; Jacobs et al, 1999b), TBX2 (Jacobs et al, 2000; Lingbeek et al, 2002) although it also has activities on the p21 promotor (Prince et al, 2004) and CBX7 (Gil et al, 2004). Importantly, only the stress induced senescence can be inhibited by all of these p16 repressors while hTERT expression is needed for full immortalization. The second class includes activators of p16 transcription, i.e. mainly the Ets proteins. The Ets proteins are direct targets of Ras-RAF-MEK signaling and can directly activate transcription of p16 (Ohtani et al, 2001). They therefore provide interesting candidates for RAS-induced senescence but can currently not explain the dependence of ROS.

Several attempts have been made to identify genes that repress senescence downstream of RAS either when overexpressed or inhibited. PLM was identified as upregulated in a microarray study of RAS-induced senescence and was shown to be sufficient for induction of senescence in the absence of RAS in both human and mouse cells (Bischof et al, 2002; de Stanchina et al, 2004; Ferbeyre et al, 2000; Fukuyo et al, 2004). PML can modulate the activity of both the p53 and the RB pathway (Ferbeyre et al, 2000; Pearson et al, 2000). Similarly to RAS-induced senescence, PML-induced senescence is dependent on Rb (Mallette et al, 2004) and Rb mutants that are less efficient in binding to E2F can induce senescence through induction of PML-nuclear bodies (PML-NB). This indicates that PML recaptures some of the characteristics of RAS-induced senescence and that PML might be one additional target of Rb during senescence (Fang et al, 2002).

A genetic screen for genes that overcome RAS-induced senescence in MEFs, identified hDRIL, an E2F binding protein. Both the p53/p21 and the p16 pathways were activated during rescue from RAS-induced senescence mediated by hDRIL (Peeper et al, 2002) and it was postulated that the anti RAS-induced senescence action of hDRIL was through release of E2F from Rb. It was later established that the effect may also relate to PML as hDRIL disintegrate the PML-NBs which further indicates that RAS-induced senescence is somehow connected to the PML-NBs (Fukuyo et al, 2004). However overexpression of hDRIL in human cells induced senescence probably in an oncogene manner similar to E2F and inhibition of hDRIL lead to an accumulation of PML-NB and PML induced senescence (Peeper et al, 2002). hDRIL can therefore not be used in human cells to understand the impact of PML on RAS-induced senescence and seems unlikely to be a key mediator of RAS-induced senescence although it can modulate the pathways.

A reverse genetics approach to identify genes that mediate RAS-induced senescence in rat embryo fibroblasts identified Seladin-1 (Wu et al, 2004). Originally described as a metabolic enzyme, Seladin-1 is activated by both RAS overexpression and hydrogen peroxide indicating that it could act as an oxidative stress sensor (Wu et al, 2004). Downregulation of Seladin-1 was further shown to allow bypass of senescence both in mouse and human fibroblasts (Wu et al, 2004). However, Seladin-1 activates the p53 pathway by releasing p53 from MDM2, which is surprising as the p53 pathway is not necessary for RAS-induced senescence in human cells (Wu et al, 2004). Future studies may demonstrate that Seladin-1 acts on the p16/Rb pathway and how it relates to the stress kinase p38. Regardless, these data further indicate that senescence downstream if RAS is stress dependent as it was activated by both RAS and hydrogen peroxide.

In summary there is some evidence that RAS-RAF-MEK-Ets plays a role during induction of senescence downstream of RAS but questions remain as inhibition of ROS, p38 or Seladin-1 also inhibits RAS-induced senescence. RAS-induced senescence seems likely to occur when p16 levels reach a critical level (Benanti and Galloway, 2004). This level could be achieved when both the direct RAS-RAF-MEK-Ets pathway is activated as well as the stress pathway mediated by ROS/p38/Seladin-1/p16. The PML protein is likely to act as an amplifier, but may not be necessary if the stress level is high enough. The kinetics of RAS-induced senescence supports this theory. While RAS expression is induced immediately, p38 signaling is activated together with p16 after four days (the design of the study did not allow a separation of p38 and p16 activation) and the senescent phenotype appears after seven days (Wang et al, 2002). Interestingly, overexpression of a constitutively active downstream target of p38, MKK6EE, induced senescence after 4 days (Haq et al, 2002). These data indicate that protein synthesis is needed at each step and that there is time for accumulation of ROS damage or a shift in the metabolic balance to activate p38/p16 which then needs further time to manifest the full senescent phenotype.

Regardless of the mechanism for RAS-induced senescence, the critical question is whether oncogene-induced senescence is an in vitro artifact or if it occurs in vivo. A recent report indicates that senescence is induced by activated E2F3 in vivo and that this process likely leads to inhibited tumor formation in vivo in mice (Denchi et al, 2005). However, given the results described above where human oncogene-induced senescence seems to be dependent on artificially high oxygen level (as RAS-induced senescence was inhibited at low oxygen levels), it is unsure if oncogene-induced senescence is an in vivo response in humans as well.

VI. Why did you senesce?

All primary human cells enter senescence after a certain number of cell divisions (Hayflick and Moorhead, 1961; Hayflick, 1965). However, senescence is defined by a set of shared characteristics, not by a common pathway. Initially, cell hybrid studies of immortal cell lines indicated that there were four different complementation groups based on the occurrence of recessive genes that could induce senescence (Tominaga et al, 2002). The chromosomes carrying three of the recessive genes responsible for three of the groups have been identified but only one gene, MORF4 has been validated as responsible for the complementation phenotype (for a detailed review see (Tominaga et al, 2002)). During the last couple of years it has become clear that primary cells from different species and cell types differ in how they induce senescence. From a simple perspective, each human cell can be described as entering senescence with p16/Rb or p53/p21 activation. This view of senescence comes from a few studies of senescence in single cells. The initial finding was that there was no gradual increase in p21 expression in human fibroblasts as reported previously (Stein et al, 1999), rather the increase was abrupt in the single cell (Herbig et al, 2003). The gradual increase is clearly a function of heterogeneous telomere lengths in a multi- cellular population, that results in slightly different replicative potential of different cells (Martin-Ruiz et al, 2004). Similarly it was described that although senescing fibroblasts can show an increase in both p16 and p53/p21 activity, these pathways are typically not active in the same cell and telomeres induce senescence exclusively by activating the p53/p21 pathway (Herbig et al, 2004). However, all data do not agree with this view as e.g. cells with a mutant TRF2, that generate telomere dysfunction before any stress induced senescence should be active, entered senescence with elevated p16 and p53 activity and abrogation of senescence was only possible with both E6 and E7 expression (Smogorzewska and de Lange, 2002). However, it can not be excluded that the TRF mutant model simultaneously induced a stress response and hence the need for both Rb and p53 inactivation, or that the appearance of stress induced senescent cells occurs very early.

The single cell studies indicate that senescence resulting from the telomere pathway and from culture stress can coexist in a population of cells. Therefore, one can define each cell type based on whether they are more likely to enter senescence as a result of culture stress or telomere erosion. The culture stress senescence appears to be similar to the senescence response driven by RAS and is characterized by p16 overexpression. Human epithelial cells (Brenner et al, 1998; Jarrard et al, 1999; Romanov et al, 2001; Schwarze et al, 2001) and keratinocytes (Munro et al, 1999) senesce with high levels of p16 but with long telomeres, and telomerase did not overcome senescence (Kiyono et al, 1998). In contrast, human fibroblasts senesce mainly because of telomere shortening with p53/p21 induction. This probably reflects that human fibroblasts are more resistant to culture stress and therefore reach the telomere restriction point and activation of ATM-p53-p21 signaling (Brown et al, 1997; Wei et al, 2001, 2003; Herbig et al, 2004). However, some fibroblast strains are also sensitive to culture stress and show a substantial stress induced senescence with p16 induction (Stein et al, 1999; Itahana et al, 2003; Bond et al, 2004; Brookes et al, 2004; Taylor et al, 2004). Further, human fibroblasts can be forced to senescence from stress (Munro et al, 2001) and can also enter a senescence state characterized by an increase in p16 expression if the telomere driven senescence is inhibited (Bond et al, 1999). These data indicate that human fibroblasts can enter stress induced senescence similar to epithelial cells under some conditions, but normally senescence from telomere signaling.

Mouse cells do not senescence because of telomere erosion as they have substantially longer telomeres (Kipling and Cooke, 1990) and can grow indefinitely in low oxygen (Busuttil et al, 2003; Parrinello et al, 2003) or low serum conditions (Woo and Poon, 2004). In contrast to human cells that senesce from culture shock, both p53 and the p16/Rb pathway are necessary, but not p21. The TRF2 mutant senescence model support that the basic mechanisms differ between mouse and human cells as mouse senescence induced by telomere damage was not dependent of p16 expression whereas human senescence was (Smogorzewska and de Lange, 2002). In summary, depending on cell type and species, the mechanisms for induction of senescence varies but importantly the end point is similar in terms of phenotypic characteristics and gene expression signatures (Larsson et al, 2004) (Figure 3).

VII. Why is senescence sometimes irreversible?

There seems to be a fundamental difference in the degree of irreversibility depending on which pathway that triggers senescence. Human fibroblasts that senesce with p16 activity do not reenter the cell cycle after microinjection of SV40 large T antigen whereas cells that senesced with p53/p21 activity do (Beausejour et al, 2003). This effect could be a result of an establishment of senescence associated heterochromatin foci (SAHF) that was described in senescent cells with an active p16/Rb pathway (Narita et al, 2003). SAHF leads to a stable repression of E2F target genes such as cyclin-A and cyclin-E (Narita et al, 2003). The mechanism could include BRG1, HDAC1, SUV39H1 and/or a transcriptionally repressive form of the histone protein H2A called macroH2A. All these proteins mediate changes on chromatin structure and have been linked to formation of SAHA or to the growth restrictive activities of Rb. BRG1 is a component of the SWI/SNF chromatin remodeling complex that can induce senescence in cells with functional Rb (Dunaief et al, 1994) and seems important for Rb mediated growth arrest (Strobeck et al, 2000) although some recent data indicate that the effects may not be directly through the physical interaction with Rb as BRG1 can induce p21 as well (Kang et al, 2004). Similarly, HDAC1 was found in complex with Rb and is also important for Rb mediated repression of cyclin-E but not necessary for Rb/SWI/SNF mediated repression of cyclin-A (Zhang et al, 2000). SUV39H1 associates with RB and corporate to repress cyclin-E probably through methylation of histone H3 followed by binding of HP1 to the chromatin and establishment of heterochromatin (Nielsen et al, 2001). MacroH2A is enriched in SAHF and could affect the chromatin structure by removing the chromatin modifications (Zhang et al, 2005). Two chaperone proteins that can assemble macroH2A onto DNA (HIRA and Asf1a) are sufficient and necessary for establishment of SAHF and senescence at least in some cell types (Zhang et al, 2005). Interestingly there could be a role of the PML-NB as the proteins that localized to SAHF first associate with the PML-NB (Zhang et al, 2005). It is likely that some of these factors contribute to stably repress E2F target genes and thereby mediate the genetic death that is characteristic of some forms of senescence. An interesting question is why only the p16/Rb pathway leads to irreversibility and not the p53/p21 pathway. It could be related to the phosphorylation pattern of Rb which will differ if mainly CDK4/cyclin-D or CDK2/cyclin-E is inhibited by p16 or p21 respectively.

VIII. Senescence and cancer

Cancer cells proliferate beyond the normal point of replicative senescence and thus need to maintain telomere lengths to continue to divide. 90% of all tumors maintain stable telomeres by overexpression of telomerase while the remaining 10% use an alternative mechanism of telomere maintenance that involves recombination, called ALT (Shay, 1997). Although this suggests that a mechanism that induces senescence in vitro is also needed for extensive proliferation in vivo; the detailed mechanisms in vitro and in vivo may not be the same. However, an interesting p53 mutant that is unable to induce apoptosis while still able to induce cell cycle arrest (at an intermediate level between wild type and p53-null), has provided some indications that senescence could operate in vivo and restrict cancer progression. Double mutants for this p53 allele did not develop early tumors compared to a p53 null mice and the tumors that eventually occurred were diploid showing that cell cycle arrest (and possibly senescence) is occurring in vivo (Liu et al, 2004). This remains the strongest principal proof for senescence being a natural mechanism that counteracts tumor progression. There are indications that senescence is a response to ongoing chemotherapy treatment, as most cell lines are able to respond to doxorubicin by induction of senescence (Chang et al, 1999) and senescence can be an in vivo response to chemotherapy in mice (Chang et al, 1999; Schmitt et al, 2002).

Could induction of senescence be a reasonable strategy for cancer treatment? There is data supporting this idea: Inhibition of telomerase by expression of a mutated telomerase RNA-component, inhibited proliferation of human cancer cells (Kim et al, 2001). Induction of senescence was also achieved by adding single stranded oligo-nucleotides (telomere repeat) to the medium of tumor cells (Li et al, 2003), although a similar treatment has also been described to induce apoptosis in another cell type (Eller et al, 2002). The mechanism involves disruption of the telomere structure, possibly by titration of some of the telomere binding proteins, and induction of a DNA damage response (Eller et al, 2003; Li et al, 2004). Interestingly the effect is specific for the telomeric repeat sequence (Li et al, 2003) and dependent on both the p53 and the Rb pathway, indicating that it probably mimics a normal replicative senescence response as well as a general stress response (Li et al, 2004). A compound was recently reported to reduce the levels of telomerase indirectly, induce senescence in vitro and showed in vivo effects in a mouse model (Incles et al, 2004; Burger et al, 2005).

While limiting cancer cell growth by induction of senescence sounds like a good strategy, it is not entirely clear how beneficial this would be in vivo. Indeed it has been discovered that senescent cells can actually promote tumor cell growth, in vitro and in vivo, in a model with pre-malignant epithelial cells (Krtolica et al, 2001; Parrinello et al, 2005). Similar promotion of cancer progression, by one cell type influencing another, has been shown in a system where fibroblasts deficient in TGF-b signaling were able to promote cancer progression in adjacent epithelial cells (Bhowmick et al, 2004). The mechanisms were described to be mediated both by cell-cell interactions as well as paracrine stimulation (Bhowmick et al, 2004; Krtolica et al, 2001). Also, senescent cells have previously been described to secrete growth factors; and media from senescent cells can be mitogenic and anti-apoptogenic (Chang et al, 2000b). In that sense the ability to enter senescence may be beneficial for overall tumor survival. An increased knowledge of senescence may therefore lead to a reevaluation of the potential for senescence as a treatment strategy and possibly show that specific inhibition of senescence, with retained apoptosis induction, during treatment with standard chemotherapy is the way forward.

IX. Senescence and aging

When Hayflick discovered that primary cells in vitro have a finite life span and enter senescence, one of the first theories that arose was that cells in a tissue would behave similarly and cause aging (Hayflick and Moorhead, 1961; Hayflick, 1965). According to the theory, these senescent cells have lost their original function and impair organ function. The aging phenotype was therefore the sum of all malfunctions in all organs that the senescent cells cause. While plausible and accepted among many researchers, the data is not convincing:

The theory dictates that the number of senescent cells increases with age in tissues and several attempts have been made to detect such an increase. Initial studies established cultures of primary cells from differently aged donors and measured their replicative life span. Some studies managed to find a decreased replicative lifespan from older subjects while others did not (Martin et al, 1970; Cristofalo et al, 1998). However this approach may not be valid as there will be a clonal expansion of the cells with the longest telomeres and although there are senescent or close to senescent cells in the population, these could be difficult to detect. Another approach is to look for a decrease in telomere lengths as a function of life span and take this as an indication of a replicative decline in the tissue. When combining all such efforts the conclusion was that although the main differences in telomere lengths depend on the individual, there is a gradual decrease of the telomeres with age in some organs (Takubo et al, 2002).

Neither of these approaches demonstrates that the senescent cells actually accumulate in a tissue. Therefore, several attempts have been made to identify an increase in senescent cells with age but the main setback of this approach has been the lack of markers for senescence, except the commonly used SA-bGAL. An increase in SA-bGAL staining cells with age has been detected in human skin (Dimri et al, 1995) and in mouse kidney (Krishnamurthy et al, 2004). In the human study only cells close to the hair follicle were stained, while the whole kidney stained blue in the mouse study, which would indicate that almost all cells in the kidney were senescent. An alternative explanation in both these studies is a lack of specificity of SA-bGAL that has been described (Severino et al, 2000). SA-bGAL staining cells have also been observed in mice after chemotherapy treatment (Schmitt et al, 2002), after liver hepactomy in third generation telomerase-activity deficient mice (Satyanarayana et al, 2003) and in a knock-out mouse for Bub1 that shows an accelerated aging phenotype (Baker et al, 2004).

Therefore, if one believes that SA-bGAL is a valid marker for senescence in vivo there seems to be evidence for senescent cells in vivo of the mouse but not humans as the age dependent increase of SA-bGAL cells in skin (Dimri et al, 1995) could not be confirmed (Severino et al, 2000). Interestingly both humans (Cawthon et al, 2003) and worms (Joeng et al, 2004) with longer telomeres show extended life span. The human telomeres were measured from blood samples and the extended life span may at least partly reflect the immune system, as the increased mortality with short telomeres was attributed by the authors to death in infectious disease and increased heart disease. The extended life span in worms was somehow related to the main survival pathway in C. elegans controlled by DAF-16.

The best link between senescence and aging comes from a premature aging syndrome called Werner syndrome. Werner syndrome patients die at an median age of 47 with myocardial infarctions and cancer and show several signs of accelerated aging (Martin, 2005). The Werner syndrome arises as a consequence of mutations of the Werner protein which is a RecQ DNA helicase (Gray et al, 1997). Interestingly, primary fibroblasts from Werner patients senesce early and show similar expression patterns as normal senescent cells indicating that the early senescence may drive an accelerated aging phenotype (Ly et al, 2000). Several lines of evidence indicate that the early senescent phenotype is related to an inability to maintain the correct telomere structure/length and that this causes the aging ohenotype as the Werner cells can be rescued from senescence by overexpression of telomerase (Wyllie et al, 2000); a third generation telomerase activity deficient mouse with a Werner mutation shows an accelerated aging phenotype (Chang et al, 2004); the Werner protein is associated with the telomere and can bind to TRF2 (Opresko et al, 2002, 2004); and cells with the Werner mutation lose their lagging strand telomere at a high rate (Crabbe et al, 2004). It has also been reported that the average telomere length in Werner cells is not different from normal cells (Schulz et al, 1996) but that could be explained by the observation that some lagging strand telomeres are very short while others are normal (Crabbe et al, 2004). Several other functions of the Werner protein has been reported including transcription (Balajee et al, 1999) but it seems likely that it is the function related to the telomere structure that drives the early senescent phenotype and possibly also the early aging phenotype. There is no data suggesting that Werner patients have more senescent cells in their tissues but given the functions of the Werner protein the effects could be in stem cell compartments that would be depleted from replicatively competent cells.

In summary there is no substantial data showing that senescence drives aging or is accumulated as a function of age in humans. Senescent cells have been detected during aging as well as in an accelerated aging phenotype and some other conditions in mice, indicating that they could exist. Interestingly gene expression data supports this species difference as the senescence transcriptome was found to be similar to that of mouse but not human aging (Wennmalm et al, in preparation).

X. Senescence vs. apoptosis

As described above, replicate senescence involves a DNA damage response that is also capable of promoting apoptosis; so why is senescence the outcome of telomere instability? The first thing to point out is that there is little evidence of senescence in human tissues, so far senescent cells have only been detected in skin of elderly people (Dimri et al, 1995), a finding that could not be repeated (Severino et al, 2000).

One obvious mechanism that could regulate the choice between senescence and apoptosis is if the apoptotic process was inhibited and senescence occurred instead as a default mechanism. The mitochondrial anti-apoptotic protein Bcl-2 has been proposed to represent such a mechanism. Unexpectedly, overexpression of Bcl-2 has been shown to induce senescence, judged by SA-bGAL staining, yet this may more resemble quiescence as p27 was overexpressed (Crescenzi et al, 2003). Bcl-2 can also accelerate RAS-induced senescence to some extent (Tombor et al, 2003). In support of the hypothesis, Bcl-2 has been described to shift the response from apoptosis to senescence when artificially overexpressed in rat cells (Rincheval et al, 2002). In the report describing a shift from apoptosis to senescence upon Bcl-2 overexpression, p21 was found to be overexpressed. In fact, this could be the reason for the shift from apoptosis to senescence as p21 expression after DNA damage lead to senescence while absence of p21 induction after DNA damage lead to apoptosis (Seoane et al, 2002). Similarly, apoptosis was associated with low p21 levels whereas senescence was associated with high p21 levels in a cancer cells treated with interferon-g (Detjen et al, 2003). If p21 decides if the response, downstream of p53induction, will be senescence or apoptosis, then an important question is why p53 sometimes induces p21 expression and sometimes not. Some of the regulation could be a result of the convergence of several pathways that directly regulate p21. For example both Miz-1 and CUGBP have been described to affect the transcription and translation of p21 respectively (Iakova et al, 2004; Seoane et al, 2002). It is also possible that the decision, of whether or not to induce p21, occurs at the level of p53 activation. Interestingly, the phosphorylation patterns of p53 during induction of senescence and after a DNA damage treatment leading to apoptosis seems to differ (Chehab et al, 1999; Webley et al, 2000). The question would then be what regulates the differential phosphorylation of p53 during senescence and apoptosis. Interestingly, there are some indications of how this could be achieved. It appears that a large DNA damage response leads to apoptosis while a low but persistent activation of p53 induces senescence. For example, upon hydrogen peroxide treatment both senescence and apoptosis are possible outcomes; apoptosis was associated with higher levels of p53 and low levels of p21 while senescence was associated with lower levels of p53 and higher levels of p21 (Chen et al, 2000). Similarly a TRF2 mutant that cause telomere dysfunction induced apoptosis or senescence, depending on the expression level and thereby the extent of telomere damage (Lechel et al, 2005); and substantial overexpression of p53 induced apoptosis while lower overexpression induced senescence (Macip et al, 2003). In summary, it seems like p21 and the nature of the p53 response is the major determinant whether the p53 response will induce apoptosis or senescence. The differential regulation of p21 needs to be further clarified.

Acknowledgements

I would like to thank Dr James A Timmons, Dr Christina Karlsson-Rosenthal and Dr Claes Wahlestedt for reading the manuscript.

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Review Article

Received: 10 June 2005; Accepted: 23 June 2005; electronically published: September 2005

References