Introduction

Senescence, the process of cellular aging, was initially described as the permanent cell cycle arrest that accompanied the replicative exhaustion of cultured human fibroblasts 1. It differs from quiescence, which is defined as a temporary cell cycle arrest, readily reversible in response to proliferative conditions, and is believed to play a role in both in vivo aging and cancer 2, 3, 4, 5, 6, 7, 8. Senescent cells are marked by characteristic changes in morphology, remain metabolically active and will not revert to proliferation even under promitogenic conditions 6, 9. Senescence has been heavily studied in vitro and in vivo, and while much has been learnt since Hayflick's initial observation, we are still far from fully understanding the molecular intricacies of this physiological phenomenon.

Telomere shortening, a normal physiological function that occurs during cellular duplication, is a driving force behind the activation of the senescence pathway 10, 11, 12, 13, 14. Senescence can also be triggered by stresses, such as DNA-damaging agents, oncogene activation or altered chromatin organization 15, 16, 17. These stresses all have the potential to initiate or promote neoplastic transformation, pointing to the importance of understanding senescence on a clinical level.

Senescence, being an end point to the cell cycle, has largely been investigated through the study of cell cycle regulatory genes. The retinoblastoma (Rb) family proteins together with respective E2F factors are critical in the progression and control of the cell cycle, specifically in the progression from G0 to S phase 18. Moreover, the role of Rb family proteins as critical effectors of cellular senescence has widely been discussed during recent years 19, 20, 21, 22, 23. Three proteins, p105/Rb, p107 and p130/Rbl2, constitute the Rb family and are characterized by a pocket domain involved in protein-protein interaction 24.

Unlike other Rb family member proteins, p130/Rbl2 is active in G0-arrested cells, where it forms a complex with E2F-4 18. This complex binds DNA and modulates E2F target genes 25, 26. In response to mitogenic stimuli, p130/Rbl2 is inactivated by cyclin-dependent kinases (CDKs) 25, 26. This allows E2F factors to activate E2F target genes, allowing the cell cycle to proceed (Figure 1) 27, 28. On the contrary, under cellular stresses, such as DNA damage, CDKs are inhibited by CDK inhibitors and Rb members remain active, thus arresting cell cycle progression 27, 28. While the role of p130/Rbl2 as the specific Rb member involved in senescence in response to CDK-inhibition and the functional compensation by other Rb family members in cells lacking functional p130/Rbl2 have already been discussed (Figure 1) 19, 21, 22, 23, there are now suggestions for a role of p130/Rbl2 during the initiation, or triggering, of the senescence process.

Figure 1
figure 1

Differential regulation of E2F target genes during G0-G1/S progression. In G0 and early G1 phase, the complex p130/Rbl2-E2F-4 binds to and represses E2F target genes. In late G1 and G1/S phase, E2F target genes are activated by the E2F-1 factor, which replaces the p130/Rbl2-E2F-4 complex 26. The transition of complexes is mediated by mitogenic stimuli, which activate cyclin-CDK complexes that in turn phosphorylate and inhibit Rb family members 18. Different studies have indicated that the three pRb family members exhibit a functional overlap, although not a complete redundancy, in many aspects of cell regulation. p107 and p130/Rbl2 appear to be redundant in regulating a specific set of E2F-responsive genes that are not subject to pRb/E2F-mediated repression 76, 77, 78. Interestingly, it has been shown that Rb−/− p107−/− murine embryonic fibroblasts (MEFs), although initially subject to growth inhibition, did not enter senescence but established a constant proliferation rate. Furthermore, ablation of all Rb-gene family members renders these cells virtually insensitive to growth inhibition and strongly increases their proliferation rate, indicating a predominant role of Rb family in cellular senescence 22, 23.

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In this review, we summarize the molecular process of senescence, dividing it into two separate temporal phases, and introduce our hypothesis of a novel role for p130/Rbl2 during the triggering of senescence as suggested by recent literature.

Two distinct phases in the senescence process

Senescence, a process more complex than originally thought, involves both physiological and morphological changes to the cells 6, 9. Our purpose is not to clarify all molecular mechanisms involved and studied in senescence, but to give an overview of the process, focusing on telomere signaling and the pathways involved in its response. For a more complete and detailed explanation of the entire process and all the pathways involved, we suggest three recent manuscripts by Muller et al., Fridman and Tainsky, and Cichowski and Hahn 6, 29, 30. To better understand the role of p130/Rbl2 in the regulation of senescence, we propose to divide the process into two distinct phases differing on timing and molecular pathways implied. We term the initial phase as the triggering of senescence, where cells stop dividing but remain metabolically active. This phase is characterized by a burst in the expression of p21CIP1, which then diminishes as senescence continues, both in vitro and in vivo 31, 32, 33. During this phase cells will express a characteristic senescent phenotype, which is, however, reversible 34. The second phase, which we term as the maintenance of senescence, occurs once the cells pass the critical midpoint and become irreversibly arrested 34, 35. This phase is characterized by the accumulation of p16INK4A, which has been shown to uniquely occur during late senescence 31, 32, 35, 36. Both p21CIP1 and p16INK4a are independently able to induce inhibition of the activity of cyclin-CDK complexes, thus modulating the hypophosphorylation of Rb family members and E2F target gene expression 6, 27, 32, 36, 37. We believe that the distinction between these two phases could be important, as diverse molecular pathways have been shown to be involved. Nonetheless it is significant to recognize them as phases of a unique process, acting concurrently or in response to one another. However, there are also data supporting the view that the two pathways are independently activated and regulated instead by the timing of events during senescence 38, 39. While both theories are currently valid, we propose that there exists a midpoint during senescence that divides these two phases.

The triggering of senescence: telomere shortening, p53 activation and p21CIP1 accumulation

Triggering of senescence has the role of both decreasing cellular proliferation, as well as preparing the cell to cease duplication and maintain its senescent state. The initiation of senescence is believed to be triggered, in physiological conditions, when progressive telomere shortening leads to the activation of p53 40, 41, 42. Telomere shortening is sensed as DNA damage and is recognized by ataxia telangiectasia mutated (ATM) protein 42. When ATM senses that there is DNA damage, it phosphorylates histone H2AX (γ-H2AX), which is found at the site of damage. It then recruits TRF1 and TRF2 followed by Chk1 and Chk2 38, 43, leading to the activation of p53 and senescence (Figure 2).

Figure 2
figure 2

Progressive telomere shortening leads to the activation of the senescence pathway. Short telomeres are sensed by ATM, or by ATR in ATM−/− cells 38, as DNA damage, which activates p53 (a). p53, acting as a transcriptional activator of CDKN1A gene, leads to the activation of p21CIP1 (b), which binds to cyclin-CDK complexes, blocking their enzyme activity 45, 46, 47, 48, 49, 50, 51. As a result, Rb members cannot be phosphorylated and inactivated by cyclin-CDK complexes, leading to cell cycle arrest (c) 44. Recent studies suggest that DNMTs could be implicated in the activation of the senescence process through the methylation of subtelomeric regions leading to progressive telomere shortening 52, 53, and through transcriptional repression of CDKN1A gene, modulating p21CIP1 transcription 54 (d). These contrary functional regulations could be understood as a “control mechanism”. In fact, when DNMTs are silenced and telomere shortening is compromised, the transcription of CDKN1A still leads to premature senescence in human cells 55. Benetti et al. 53 have also shown that p130/Rbl2 could have a specific role in telomere length regulation through the modulation of DNMT levels, specifically DNMT3a and DNMT3b (e).

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Cyclin-Cdk complexes are critical in continuing the progression of the cell cycle. They inhibit Rb family member proteins allowing E2F factors to bind and initiate transcription of cell cycle-promoting genes 44. The activation of p53, a critical transcription factor involved in cell cycle control pathways, leads to the transcription of CDKN1A gene and accumulation of p21CIP1 in senescent cells 45. p21CIP1 binds to and inhibits the cyclin-Cdk complexes as well as the proliferating cell nuclear antigen (PCNA) through complex formation, thus regulating cell cycle progression, DNA synthesis and DNA repair (Figure 2) 6, 32, 46, 47, 48, 49, 50, 51.

Telomere shortening continues to be a topic of great interest, given its initiating role in senescence, but much is yet to be discovered. Gonzalo et al. 52 have shown that defects in DNA methyltransferases (DNMTs) lead to decreased methylation of subtelomeric regions and elongated telomere length in mouse embryonic stem cells. Benetti et al. 53 further show that overexpression of DNMTs can rescue DNA methylation in subtelomeric regions of DICER−/− mouse embryonic stem cells. The precise role of DNMTs in the regulation of telomere length remains to be defined, but DNMTs seem to be implicated in telomere shortening as well as in the triggering of senescence 54, 55. The principal human DNMTs are hDNMT1, hDNMT3a and hDNMT3b 56, 57, 58. Zheng et al. 54 have shown that protein levels of DNMT1 and DNMT3a increase from young to middle-aged human fibroblasts but decrease in senescent fibroblasts. Moreover, in non-senescent cells DNMTs methylate the promoter of p21CIP1, repressing its transcription 54, 55.

The implication of DNMTs in the triggering of senescence, given the data presented, could be suggested by the two following scenarios (Figure 2). First, they could act as promoters of progressive telomere shortening, leading to the activation of the p53 pathway 52, 53. Second, they could act as transcriptional repressors of CDKN1A gene, modulating p21CIP1 transcription 54, 55. It is interesting to note that while the level of DNMTs increases in middle-aged human fibroblasts, it decreases in senescent fibroblasts 54, 59. This supports the role of DNMTs both in progressive telomere shortening in non-senescent cells and the subsequent activation of CDKN1A transcription in senescent cells.

During senescence, the regulation of DNMTs likely involves p130/Rbl2 (Figure 2). Benetti et al. 53 have shown that in DICER−/− mouse ES cells, where there is an increase in p130/Rbl2 transcript, there is an aberrant elongation of telomeres caused by decreased levels of DNMT3a and DNMT3b. Transfection of the miR-290 cluster, which is involved in the silencing of p130/Rbl2, has been shown to rescue DNMT levels. The direct involvement of p130/Rbl2 in the triggering of senescence is supported by an intriguing result by Lehmann et al. 60, where silencing of p130/Rbl2 in p105/Rb−/− cells led to premature senescence, while silencing of p107/Rbl1 led to resistance of irradiation-induced senescence. The specific role of p130/Rbl2, but not other Rb family proteins, in the regulation of DNMTs could explain this. It could then be hypothesized that the silencing of p130/Rbl2 would accelerate telomere shortening through deregulation of DNMTs, thus triggering senescence. These are only observations and must be confirmed through further studies; however, the role of p130/Rbl2 in telomere shortening and in the triggering of senescence is strongly suggested.

The maintenance of senescence

The maintenance of senescence is still not well defined. The main effector seems to be p16INK4a, inhibitor of Cyclin D-associated kinases 61, 62, which is able to independently induce senescence 31, 36, 63. While its exact role is not completely understood, it is believed to be involved in the maintenance of cell cycle arrest during the senescence process, because it accumulates in late senescence 32 and its expression and function are independent of telomere status 64, 65. p16INK4a maintains the activity of Rb family members by preventing cyclin D1 from forming complexes with CDKs 4 and 6, which are initially responsible for Rb family inhibition 31. Beauséjour et al. 34 have shown that proliferating cells with p16INK4a stably silenced can proceed into senescence; however, they maintain elevated levels of p21CIP1, and the subsequent suppression of p53 can completely reverse the senescent phenotype. Instead, if p16INK4a is stably silenced after the cells have entered senescence, then suppression of p53 cannot reverse the senescent phenotype.

Evidence suggests a specific role for the p16INK4a/Rb pathway in the formation of senescence-associated heterochromatic foci (SAHF), a heterochromatic DNA structure that accumulates in senescent human cells 20, 66, 67, 68. First, Rb interacts with the histone methylase, Suv39H1, which methylates lysine 9 of histone H3 (K9M-H3) and forms a complex with methyl-lysine binding protein, HP1, in the promoter region of E2F target genes, resulting in transcriptional repression, which is coincident with SAHF formation in senescence cells 20, 69, 70. In fact, SAHF, which are rich in K9M-H3 and HP1, are induced by enforced p16INK4a expression and are reduced by suppression of p16INK4a or p105/Rb 20. Second, p16INK4a/Rb cooperate with the ASF1a/HIRA pathway to drive chromosome condensation and SAHF formation 68, 71, 72.

Recently, it has been shown that once senescence becomes irreversible, there is a p16INK4a-dependent activation of reactive oxygen species (ROS), which maintain their levels through a p16INK4a-independent positive feedback mechanism 35, 73. The role of ROS in senescence remains unknown, as do many other aspects of the senescence process. However, there is now suggestion for a p16INK4a-dependent midpoint in the senescence process, which leads to both SAHF formation and activation of ROS production. Once the midpoint is surpassed, independently from p16INK4a levels, it would no longer be possible for the cell to return to “youth” (Figure 3).

Figure 3
figure 3

p16INK4a activation of maintenance of senescence. The maintenance of senescence is obtained after the cell surpasses the “midpoint”, regulated by the p16INK4a/Rb pathway 33. Once this occurs, cells cannot “go back”. It is suggested that this irreversible state is caused by two processes, both of which represent consequences of the p16INK4a/Rb pathway: the formation of irriversibly silenced senescence-associated heterochromatic foci (SAHF) 20, 66, 67, 68, 69, 70, 71, 72 and the production of reactive oxygen species (ROS) that maintain their levels through a positive feedback mechanism 73.

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Conclusion

The role of Rb family members in senescence has yet to be clearly defined. It is known that all Rb family members are implicated in the senescence process, but there are suggestions for a specific role of p130/Rbl2 that does not simply represent the compensation of p105/Rb 19, 21. Recently, it has been shown that downregulation of p130/Rbl2 by siRNA in p105/Rb−/− cells caused premature senescence 60, instead of prolonged life as would be expected if p130/Rbl2 acted in substitution of p105/Rb. To better understand the different roles of proteins that share similar structure and function, it is important to understand the interactions with their respective effector molecules. E2F-1, E2F-2 and E2F-3 can be bound by p105/Rb and are present only after mitogenic stimuli, whereas E2F-4, bound by p130/Rbl2, is much more abundant and functional in G0-arrested cells than other E2F factors 35, 74, 75. During senescence, Rb members block the transcriptional activity of E2F factors 19, 20, 21. p130/Rbl2 on the other hand, is hypothesized to play a specific role in the triggering of senescence through its regulation of DNMTs 53. The premature senescence found in p130/Rbl2-silenced cells could be explained by an excessive presence of DNMTs that could facilitate the shortening of telomeres 53, 60.

While it is understood that p130/Rbl2 plays an important role in cell cycle arrest, taking a critical look at the current literature suggests evidence supporting its unique role in cellular senescence. Here we have attempted to analyze how its role could be implicated given the current literature. Furthermore, we have discussed the evidence for a specific midpoint in senescence that determines whether the cell can revert back into the cell cycle or can only remain senescent and proceed toward cell death. Clearly, additional studies are needed to elucidate the entire senescence pathway and the specifics of p130/Rbl2 function. The senescence process includes various pathways and is indeed a complex molecular mechanism that is only beginning to be appreciated in recent years.