Replicative senescence at the molecular level in vitro

Telomere-independent senescence (TIS)

Like p21, p16 is a CDK inhibitor involved in cell cycle arrest. Over expression of p16 causes G1 arrest in early passage cells by inhibiting the phosphorylation of the retinoblastoma protein (Kato et al, 1998). However, the removal of p16 activity results in only minimal lifespan extension that was terminated by senescence (Wei et al, 2003). p16 plays a role in maintaining senescence triggered by a short telomere. It appears that p21 is the initiating factor in the growth arrest observed in senescent cells, but p16 is required to maintain that state. p21 has been shown to initially increase in fibroblast cultures but later gradually decrease (Alcorta et al, 1996). During the decline of p21, p16 protein levels gradually increased in senescent cultures, reaching nearly 40-fold higher than in early cultures.

P16 is thought to trigger senescence independently of telomere length. For example, the inactivated p53 in two different fibroblast strains: WI-38 (from foetal lung) and BJ (from neonatal foreskin) resulted in the continuation of growth without Rb inactivation in BJ cells, whereas WI-38 cells fail to grow even when both Rb and p53 are inactivated. It was suggested that these two outcomes was due to the intrinsic differences in the ability to induce p16 at senescence. This proposal was based on the understanding that like WI-38, other human cells appear to undergo replicative senescence prior to telomere shortening by the induction of p16 (Itahana et al, 2003). The ability of p16 to cause growth arrest even after Rb inactivation also suggests that p16 can function independently of Rb activity. Further support for a telomere-independent mechanism was provided from studies which have shown that telomerase activity alone was insufficient to extend the replicative potential of human keratinocytes or mammary epithelial cells (Kiyono et al, 1998). It was shown that down-regulation of p16 in combination with telomerase activity did lead to replicative lifespan extension. Another study found that cyclin D1 over-expression in primary oral keratinocytes extend the lifespan, whereas the combination of cyclin D1 over-expression and p53 inactivation led to their immortalisation (Opitz et al, 2001). Cyclin D1 forms complexes with CDK4 which subsequently phosphorylates and inactivates retinoblastoma (Rb) growth repression (Connell-Crowley et al, 1997). P16 inhibits cell cycle progression by inhibiting CDK4. It appears that the over-expression of cyclin D1 bypasses p16-TIS resulting in an extension of lifespan. In this instance the p16 mechanism has become redundant and the cells continue to divide until TDS mechanism is activated. This may be why these cells are immortalised by p53 inactivation.

This telomere-independent mechanism may be the primary trigger of cellular senescence in mice and rats. Mice and rats have telomeres which are 5-10 times longer than those of human cells (Shay and Wright, 2001). Despite this increase in length, rodent cells engineered to lack telomerase show telomere shortening with no effect on replicative potential. These cells senesce in the presence of long telomeres. Since mouse and rat cells repair DNA damage far less efficiently than do human cells, it has been suggested that such damage may be the trigger for p16 induction.