Replicative senescence at the molecular level in vitro

Telomere-dependent senescence (TDS)

Telomere attrition is thought to be the predominant mechanism which ultimately leads to a cell becoming senescent, caused by the inability of polymerase to completely replicate DNA at the 5’ end. The presence of a short telomere is thought to disrupt a protective telomeric structure, exposing loose ends of DNA which trigger a DNA damage response and consequently causes a cell to enter cellular senescence.

Human telomeres consist of short tandem GC-rich repeats (TTAGGG/AATCCC). These telomeric repeats are predominantly double-stranded with a 3’ single-stranded overhang terminating with a G-rich tail (Collins, 2000). The proposed mechanism by which telomeres and their associated proteins protect the chromosome ends is by creating large terminal loops known as a t-loop (Greider, 1999). In this structure, the double-stranded telomeric DNA is looped around and the 3’ single-strand overhang invades the duplex of telomeric repeats and forms a displacement loop (D-loop) structure is important for maintaining chromosomal stability. Without this protection, it is thought that the ends of chromosomes would become exposed, resembling double-strand breaks which can trigger cell-cycle arrest and senescence (Shore, 2001).

Telomeres can be elongated by adding telomeric repeats to the 3’ end of DNA. This can be carried out by telomerase, an enzyme which consists of an RNA molecule and a catalytic component known as hTERT. Telomerase is a reverse transcriptase which uses its RNA component as a template to reverse transcribes DNA back to the ends of chromosomes. Telomerase activity is repressed in most somatic tissue and reactivated in ~90% of human cancers (Artandi, 2006). Introduction of telomerase into normal somatic cells has been shown to extend replicative life-span (Bodnar et al, 1998) and not induce changes associated with a malignant phenotype (Jiang et al, 1999). Since telomerase is not present in somatic cells, they progressively get shorter.

The majority of short-telomere growth arrested cells is thought to occur in the G1 phase of the cell cycle, a process regulated by proteins such as p21. The appearance of DNA damage results in the activation of p53, a transcription factor whose activation results numerous downstream responses including the up-regulation of p21 and activation of DNA repair proteins. P21 is a cyclin dependent kinase (CDK) inhibitor which halts cell cycle progression by directly interacting and inhibiting cyclin-CDK complexes and consequently preventing the phosphorylation of Rb.

Experiments in which p21 was inactivated within senescent cells resulted in the ability of these cells to re-enter the S phase of the cell cycle, but were still unable to divide (Ma et al, 1999). It was concluded that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 is insufficient to permit cells to complete the cell cycle. Since p21 expression has been shown to be dependent upon p53, the impact of p53 inactivation on cell cycle progression was also investigated (Bond et al, 1999). Fibroblasts lacking p53 function bypassed replicative senescence and continued to proliferate for another 20 CPD before entering growth arrest. The point at which growth arrest occurs due to replicative senescence is also known as M1, whereas the second growth arrest point if replicative senescence is bypassed is known as M2 or crisis. Cells which enter crisis result in apoptosis rather than senescence. This suggests that when a cell becomes senescent at M1 it may not be due to the disruption of the telomere-end structure. Since there appears to be a reserved replicative capacity, it may only be at M2 that the telomere end-structure becomes disrupted, and this may be the reason why apoptosis is common at this point. The idea that a cell becomes senescent long before the telomere-end structure is disrupted suggests that telomere length may not be the trigger for replicative senescence and instead is due to another mechanism.

One such mechanism may be alterations or exposure of the telomere 3’ single-stranded overhang. One study reported that exposure of telomere 3’ overhang sequences induces senescence (Li et al, 2003). In this study, cells were exposed to oligonucleotides homologous to the telomere 3’ overhang sequence TTAGGG for 1 week. As a result, a senescent phenotype in cultured fibroblasts was observed. The team concluded that exposure the 3’ overhang by t-loop disruption or possibly DNA damage leads replicative senescence. Telomere shortening may result in the exposure of the 3’ overhangs, but the telomere end-structure is still maintained efficiently to prevent complete disruption. Interestingly, this 3’ overhang has been found to be eroded at replicative senescence indicating it as a possible trigger (Stewart et al, 2003). It was indicated that overhang erosion is the result of continuous cell division and not a consequence of senescence. Therefore it was concluded that this alteration in telomere structure and not overall telomere length serves as a trigger for replicative senescence.