Heterogeneity in Senescence

One of the problems with the idea of telomeres working as a clock is that the Hayflick limit is a fixed number only with respect to large populations, that is, in a stochastic sense. In contrast, individual cell lineages vary greatly in their replicative potential.

Smith and Hayflick (1974) showed large variations in the population doubling potential of fibroblast clones isolated from WI-38 and WI-26 cell cultures (Smith and Hayflick 1974). This finding raised concerns over studies involving mass cultures due to heterogeneous populations containing cells at different population doubling levels.

Later, Smith and Whitney, in a classic experiment, showed heterogeneity in doubling potential of individual cells from clonally derived populations (Smith and Whitney 1980). Thus it was suggested that heterogeneity could not be explained solely by the differences between individual cells' life histories. Rather, stochastic factors must be important to determine the replicative potential of cells.

Other studies showed that the fraction of cells able to divide decreased progressively with increasing population doublings, using BrdU labeling (Kill et al. 1994), Ki67 staining (Thomas et al. 1997), and p53-reporter assay (Bond et al. 1994). It was also shown that the percentage of cells stained positive for y-H2AX, a marker for senescence-associated DNA damage foci that might be formed in response to functionally uncapped telomeres, increased with population doubling level (Fagagna et al. 2003).

Thus, life span heterogeneity can be characterized by the fraction of cells displaying a senescent phenotype. However, the question remains, what causes this heterogeneity?

It is often assumed that cells that lose their division capacity early do so by a process termed premature senescence, which is a telomere-independent cellular response to a variety of stresses. Premature senescence can in fact be induced by a variety of experimental conditions:

Human epithelial cells encounter a telomere-independent, p16-dependent growth arrest in response to suboptimal culture conditions (Stampfer and Yaswen 2003). Overexpression of oncogenes, such as activated RAS or RAF, can induce a senescence-like arrest in primary human or mouse cells (Ferbeyre et al. 2002, Lin et al. 1998, Serrano et al. 1997), and this is dependent on p16 (Benanti and

Galloway 2004). Modification of chromatin by inhibitors of histone deacetylases also induces a senescent phenotype (Ogryzko et al. 1996). Oxidative stress, ionizing radiation, and other DNA-damaging agents can also induce the senescent phenotype without any detectable telomere shortening (Dumont et al. 2000, Herskind and Rodemann 2000, Jeyapalan et al. 2004, Robles and Adami 1998). Therefore, it is sometimes assumed that all early-occurring senescence might be both stress-induced and telomere-independent and thus premature. In other words, in any given cell culture experiment, most cells would senesce by a process that is altogether different from telomere-driven senescence, which would happen only in the last surviving clone. However, telomere shortening is by itself distinctly stress-dependent (von Zglinicki 2002), suggesting an alternative explanation for cell-to-cell heterogeneity in replicative senescence.

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