Progress paper
Regulation of growth arrest in senescence: Telomere damage is not the end of the story

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Abstract

After a limited number of divisions, most eukaryotic cells grown in culture will undergo a terminal growth arrest called cellular senescence. This growth arrest is thought to be a consequence of progressive telomere shortening that occurs due to incomplete DNA replication of the chromosome ends. In addition, cellular senescence can also be induced by a number of environmental stresses and signaling imbalances which are independent of telomere shortening. The cyclin dependent kinase inhibitors p21 and p16INK4a have been shown to execute and maintain the cell cycle arrest in senescence but the nature of the signals that cause upregulation of these inhibitors in senescent cells are only now starting to be discovered. Here we will review the current literature that leads us to propose a model how independent signals activate distinct signaling pathways to regulate p21 and p16INK4a levels in senescent cells.

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.

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