Progress paperRegulation of growth arrest in senescence: Telomere damage is not the end of the story
Introduction
With the exception of a few cell types such as stem and germ line cells, all human somatic cells have a limited proliferative lifespan. A detailed understanding of how cells limit their replicative potential is of critical importance since the mechanisms restricting proliferation of cells also prevent tumorigenesis. This is supported by the observation that expression of several oncogenes in primary human cells cause a senescence-like cell cycle arrest, and that mutations in certain tumor suppressor genes compromise the senescence arrest (de Stanchina et al., 1998, Serrano et al., 1997, Zhu et al., 1998, Zindy et al., 1998). Although it has not been determined experimentally, it is believed that by limiting the number of divisions a cell can undergo, organisms prevent the accumulation of cells that have acquired numerous, potentially transforming mutations (Campisi, 2001).
Normal cells grown in culture undergo senescence after a finite number of population doublings, and in response to a variety of stresses and signaling imbalances. The limited replicative potential of cells was first described in 1961 (Hayflick and Moorhead, 1961) but it took another decade to realize the mechanisms underlying this phenomenon. Shortly after the discovery of the semi-conservative nature of DNA replication, it was recognized that the very 3′ ends of linear chromosomes could not be copied by the DNA replication machinery because the RNA primer, which initiates de novo DNA synthesis, is removed during Okazaki fragment processing (Olovnikov, 1973). This predicament gave rise to the idea that cells lose a short segment of DNA from the 5′ end of each newly synthesized daughter strand every time the cell replicates its chromosomes. Since human chromosomes end in 5–15 kb of non-coding, repetitive DNA called telomeres, such progressive loss would not compromise the integrity of the genome until a critical degree of shortening is reached. Replicative senescence is believed to be triggered once this critical telomere length is surpassed. In addition to replication-induced loss of telomeric DNA, other factors such as exonucleases that process the 5′ end of the parental strand (Lydall, 2003), and reactive oxygen species (ROS) (von Zglinicki, 2002) have also been suggested to contribute to the shortening of telomeric DNA in eukaryotic cells. A number of studies have demonstrated a strong correlation between the replicative age of cells growing in culture, or in intact tissue (Friedrich et al., 2000, Harley et al., 1990, Hastie et al., 1990) and telomere length, however, whether telomere shortening is indicative of replicative age in living organisms is still unclear (Cristofalo et al., 2004).
Human telomeres consist of repetitive duplex TTAGGG repeats that are terminated by a 100–400 nucleotide-long 3′-single strand overhang (Ben-Porath and Weinberg, 2004, Makarov et al., 1997, Wright et al., 1997). This overhang invades the double stranded-telomeric DNA and, together with a number of proteins including TRF1, TRF2 and Pot1, forms a circular structure called the t-loop (de Lange, 2002, Griffith et al., 1999). The t-loop prevents telomeres from being recognized as broken chromosome ends, thereby suppressing the activation of DNA damage checkpoint signaling cascades (de Lange, 2002). A DNA damage response originating at telomeric DNA sequences has recently been observed in un-manipulated human fibroblasts that had been passaged to senescence (d’Adda di Fagagna et al., 2003, Gire et al., 2004, Herbig et al., 2004, Sedelnikova et al., 2004, Zou et al., 2004). Together, these studies strongly support a long-standing hypothesis that replicative senescence is a response to short dysfunctional telomeres that, once unprotected, activate a DNA damage response resulting in permanent cell cycle arrest.
Section snippets
Mediators of replicative senescence
Progression through the eukaryotic cell division cycle is in large part controlled by the activity of the cyclin dependent kinases (CDK). In G1 phase of the cell cycle, CDK4 and CDK6 become activated upon binding to the D-type cyclins, whereas cyclins E and A activate CDK2. Both, cyclinE-CDK2 and the D type cyclin-CDK4/6 complexes phosphorylate the retinoblastoma protein (Rb), whose primary function is to sequester, and thereby inactivate, several members of the E2F family of transcription
Upregulation of p21 in senescence
Levels of p21 are regulated by transcriptional activation as well as by post-transcriptional (de)stabilization of mRNA and protein. Transcription factors that are known to increase p21 mRNA levels are p53, Sp1, Sp3, E2Fs, STATs, and AP2 among others (Gartel and Tyner, 1999). p21 upregulation has been observed in a variety of cellular environments and in response to a number of stress- and signaling-molecules, however, the transcriptional activation of the p21 gene in response to DNA damage and
Upregulation of p16INK4a in senescence
p16INK4a is upregulated in response to hypermitogenic, potentially oncogenic signals, such as those elicited by overexpression of RAS, MAP kinases or Myc (Drayton et al., 2003, Lin et al., 1998, Serrano et al., 1997, Zhu et al., 1998). In addition, p16INK4a levels increase due to non-optimal tissue culture conditions (Ramirez et al., 2001), senescence (Alcorta et al., 1996, Hara et al., 1996), and in response to DNA damage induced either by drugs (Robles and Adami, 1998) or by a dominant
Cooperation of p21 and p16INK4a in senescence
Initial observations that p21 levels peak in early senescent cells but decline again at later times when p16 levels are at their maximum led to the hypothesis that p21 initiates the senescence program by temporarily arresting cells and that p16 maintains the cell cycle arrest of senescent cells (Alcorta et al., 1996, Hara et al., 1996, Stein et al., 1999). Since these experiments were performed by immunoblotting of mass cultures, they did not take into account that cells grown in tissue culture
Conclusions
A detailed understanding of the mechanisms that limit the replicative potential of human cells has been emerging in recent years from a number of studies that have dissected the individual contribution of different stress signaling pathways to the senescence arrest. Evidence has been accumulating that multiple independent pathways cooperate in aging human cells to induce a cell cycle arrest that prevents the accumulation of mutations which might have disastrous consequences for an organism.
References (98)
- et al.
Investigation of the role of G1/S cell cycle mediators in cellular senescence
Exp. Cell Res.
(1993) - et al.
Direct evidence from siRNA-directed “knock down” that p16(INK4a) is required for human fibroblast senescence and for limiting ras-induced epithelial cell proliferation
Exp. Cell Res.
(2004) Cellular senescence as a tumor-suppressor mechanism
Trends Cell Biol.
(2001)- et al.
Replicative senescence: a critical review
Mech. Ageing Dev.
(2004) - et al.
Tumor suppressor p16INK4a determines sensitivity of human cells to transformation by cooperating cellular oncogenes
Cancer Cell
(2003) - et al.
Telomere length in different tissues of elderly patients
Mech. Ageing Dev.
(2000) - et al.
Transcriptional regulation of the p21(WAF1/CIP1) gene
Exp. Cell Res.
(1999) - et al.
Mammalian telomeres end in a large duplex loop
Cell
(1999) - et al.
Cooperative effect of antisense-Rb and antisense-p53 oligomers on the extension of life span in human diploid fibroblasts, TIG-1
Biochem. Biophys. Res. Commun.
(1991) - et al.
The serial cultivation of human diploid cell strains
Exp. Cell Res.
(1961)
Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a)
Mol. Cell
Replicative senescence of human fibroblasts: the role of Ras-dependent signaling and oxidative stress
Exp. Gerontol.
Reinitiation of host DNA synthesis in senescent human diploid cells by infection with Simian virus 40
Exp. Cell Res.
Progress of aging in human diploid cells transformed with a tsA mutant of simian virus 40
Exp. Cell Res.
A role for p53 in maintaining and establishing the quiescence growth arrest in human cells
J. Biol. Chem.
Significant role for p16INK4a in p53-independent telomere-directed senescence
Curr. Biol.
A self-enabling TGFbeta response coupled to stress signaling: smad engages stress response factor ATF3 for Id1 repression in epithelial cells
Mol. Cell
Cytoplasmic retention of p-Erk1/2 and nuclear accumulation of actin proteins during cellular senescence in human diploid fibroblasts
Mech. Ageing Dev.
BJ fibroblasts display high antioxidant capacity and slow telomere shortening independent of hTERT transfection
Free Radic. Biol. Med.
Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening
Cell
Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts
Curr. Biol.
Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence
Cell
Cloning of senescent cell derived inhibitors of DNA synthesis using an expression screen
Exp. Cell Res.
A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon
J. Theor. Biol.
Mitogen-activated protein kinase pathways
Curr. Opin. Cell Biol.
Oncogenic functions of tumour suppressor p21(Waf1/Cip1/Sdi1): association with cell senescence and tumour-promoting activities of stromal fibroblasts
Cancer Lett.
Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a
Cell
A role for both Rb and p53 in the regulation of human cellular senescence
Exp. Cell Res.
Altered composition and DNA binding activity of the AP-1 transcription factor during the ageing of human fibroblasts
Mech. Ageing Dev.
DNA damage foci at dysfunctional telomeres
Curr. Biol.
Lack of Elk-1 phosphorylation and dysregulation of the extracellular regulated kinase signaling pathway in senescent human fibroblast
Exp. Cell Res.
Correlation between the presence of T-antigen and the reinitiation of host DNA synthesis in senescent human diploid fibroblasts after SV40 infection
Exp. Cell Res.
TRF2 protects human telomeres from end-to-end fusions
Cell
Cell-cycle inhibitors: three families united by a common cause
Gene
Oxidative stress shortens telomeres
Trends Biochem. Sci.
Involvement of the cyclin-dependent kinase inhibitor p16(INK4a) in replicative senescence of normal human fibroblasts
Proc. Natl. Acad. Sci. U.S.A.
Increased activity of p53 in senescing fibroblasts
Proc. Natl. Acad. Sci. U.S.A.
Reversal of human cellular senescence: roles of the p53 and p16 pathways
EMBO J.
When cells get stressed: an integrative view of cellular senescence
J. Clin. Invest.
Spontaneous abnormalities in normal fibroblasts from patients with Li-Fraumeni cancer syndrome: aneuploidy and immortalization
Cancer Res.
Evidence that transcriptional activation by p53 plays a direct role in the induction of cellular senescence
Oncogene
Escape from senescence in human diploid fibroblasts induced directly by mutant p53
Oncogene
INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence
EMBO J.
Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts
Science
A DNA damage checkpoint response in telomere-initiated senescence
Nature
Protection of mammalian telomeres
Oncogene
E1A signaling to p53 invovles the p19ARF tumor suppressor
Genes Dev.
Downregulation of 14-3-3sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes
J. Cell Biol.
A biomarker that identifies senescent human cells in culture and in aging skin in vivo
Proc. Natl. Acad. Sci. U.S.A.
Cited by (143)
Cellular senescence
2022, Cellular Senescence in DiseasemTOR as a senescence manipulation target: A forked road
2021, Advances in Cancer ResearchCitation Excerpt :Interestingly, most of the molecular hallmarks of aging are also found in senescent cells suggesting that these cells represent a particularly relevant target to modulate age-associated diseases (Hernandez-Segura et al., 2018; Rodier & Campisi, 2011). Senescent cells have been shown to accumulate with age in tissues of rodents, primates and humans (Dimri et al., 1995; Herbig & Sedivy, 2006; van Deursen, 2014; Wang et al., 2009). In rodent models, it is clear that senescent cells contribute in part to the deterioration of tissue homeostasis associated with age-associated diseases and aging.
Telomeres and genomic instability during early development
2020, European Journal of Medical GeneticsThe impact of cerebrovascular aging on vascular cognitive impairment and dementia
2017, Ageing Research ReviewsCitation Excerpt :These detrimental factors are likely to function as paracrine mediators, altering neighboring cells and modulating the internal milieu. Senescent cells are known to accumulate with age in a variety of tissues (Erusalimsky and Kurz, 2005; Herbig and Sedivy, 2006; Jeyapalan et al., 2007; KISHI, 2004). Studies on cultured endothelial cells suggest that oxidative stress is a major stimulus to induce endothelial senescence (Erusalimsky, 2009).