Relationship between replicative capacity and organismal ageing

Leonard Hayflick was the first to propose that the senescence of normal cells may contribute to the organismal ageing. Investigations into this proposal started by comparing the replicative potential of cells, usually fibroblasts, extracted from individuals at various ages.

The first of these studies showed an inverse relationship between donor age and the number of population doublings achieved in vitro (Martin et al, 1970). This study looked at the replicative lifespan of fibroblasts taken from 100 subjects with an age range from foetal to 90 years. These cells were cultured and the number of population doublings before entering senescence was recorded. The results showed that the replicative potential decreased as donor age increased. A later study showed similar results (Schneider, 1979). This study looked at the ability of fibroblasts taken from young (20-35 years) and old (65+ years) to proliferate in culture. It was reported that cell cultures from old human donors have a reduction in their proliferative capacity. A more recent study looked at the replicative capacity of human adrenocortical cells to proliferate as a function of donor age (Yang et al, 2001). Again, it was found that younger cells have a higher proliferative capability than the old. In this instance, population doubling fell from 50 for foetal cells to almost a total lack of division in culture from older cells.

To investigate the possible link between replicative lifespan and organismal ageing, a few studies compared replicative capacity with longevity in animals. One such study investigated the relationship between longevity of eight mammalian species (mouse, rat, rat-kangaroo, mink, rabbit, bat, horse and human) and the lifespan of normal fibroblasts in vitro (Röhme, 1981). It was reported that there was a direct relationship found between the longevity of the eight mammalian species and the replicative capacity of their cultured fibroblasts. A much later, but similar study, compared animal life spans and in vitro replicative capacity of skin fibroblasts in groupings of small, middle, large, and very large breeds of dogs of specific ages (Li et al, 1996). It was found that the life spans were inversely correlated to the frame sizes of the breeds. It was shown that all the small breeds studied have a longer life span than that of the large breeds. The replicative capacity of fibroblasts from the large dogs (Great Dane and Irish Wolfhound) was significantly decreased compared with that of the small dogs. The reasoning behind these observations may again be due to varying degrees of cell turnover between the species. Large dogs consist of more cells than small dogs and as a result more cell turnover was initially required in their development compared to small dogs. This increase in cell turnover would subsequently lead to a decrease in replicative potential and an increase in the rate of senescent cell formation.

Interestingly, a recent study looked at the replicative capacity of 124 skin fibroblast cell lines from donors of different ages which were medically examined and declared “healthy” (Cristofalo et al, 1998). Healthy people were used specifically as previous studies, discussed later, have shown that disease states may accelerate the reduction in replicative capacity. Results indicated that there was no significant correlation between the replicative capacity of the cell lines and donor age. In the same study, a comparison of multiple cell lines established from the same donors of different ages also failed to show any significant differences. It was concluded that the replicative capacity of fibroblasts in vitro does not correlate with donor age. However, differences in replicative capacity with age may only be observed as a result of increased cell turnover in response to disease and cellular injury. Therefore, a healthy old person who has had little or no cellular injuries or disease would have had little cell turnover and therefore have cells which may have a replicative capacity similar to someone much younger. Thus, this study supports the notion that replicative capacity is an indicator of biological age.

Replicative senescence at the cellular level in vitro

Historical overview

Until the middle of the 20th century, it was widely held that normal mitotic tissue could not age in a degenerative sense because it had an indefinite capacity to proliferate. This view had developed for two reasons. Firstly, the Nobel laureate Alexis Carrel appeared to have demonstrated the long-term cultivation of normal chick fibroblasts for periods considerably in excess of the lifetime of the animal (Parker, 1938; Witkowski, 1990). Secondly, the technical difficulties associated with tissue culture techniques until the 1950s rendered the duplication of Carrel’s studies very difficult for all but a few highly specialized laboratories (Parker, 1938). Ageing was not the primary research interest of these centres. Thus, it was not until a series of classic experiments by Hayflick & Moorhead in the early 1960s which demonstrated that cultures of normal human fibroblasts did not have an infinite capacity to expand, brought about the idea of intrinsically immortal mitotic tissue into question. Their work demonstrated that, after a finite period of growth, cultures of normal human fibroblasts became completely composed of viable but non-dividing cells (Hayflick & Moorhead 1961; Hayflick 1965). These initial observations have been reproduced in hundreds of studies, and since that time virtually all human mitotic cell types subjected to rigorous study have been shown to undergo this cellular senescence in culture.

Dynamics of normal cell populations.

The early observation by Hayflick and Moorehead (1961) that cultured cells have a maximum limit on the number of divisions before entering senescence lead to the assumption that all cells in a given culture divide roughly the same number of times before entering senescence. Hayflick considered the senescence of cultures was marked by three distinct phases. In Phase 1 the initial culture, was considered to terminate with the formation of the first confluent sheet of cells. Phase 2 was characterised by vigorous growth requiring repeated subculture. In phase 3, the senescence of the culture was characterised by the cessation of mitosis. In this model it was assumed that cultures were composed of a homogenous population of cells which were either all growing (Phase 1 or 2) or all non-growing (Phase 3) and that failure to grow was due to cell death (Kalashnik et al, 2000). The notion that senescence was cell death was soon disproved with the demonstration that RNA synthesis occurred in these cells (Macieira-Coelho et al, 1966). Evidence against the idea that cultures were composed of homogenous populations was provided by a number of different studies. Cristofalo and Scharf (1973) demonstrated the presence of senescent cells in early passage cultures using long pulse-labelling experiments on embryonic fibroblasts. 3H-thymidine labels those cells which have entered S-phase (dividing cells), and since senescence cells are halted in G1 they cannot enter S-phase, and so the percentage of unlabelled cells can be calculated. It was shown that senescent cells are present in early passage cultures and that the percentage of senescent cells gradually increases with each serial passage of the culture. This observation was explained by experiments demonstrating that cultured fibroblasts are composed of cells which display variation in proliferative potential (Smith and Whitney, 1980). Related experiments also showed that two cells arising from a single mitosis differed in their ability to proliferate by as many as eight doublings (Jones et al, 1985). Using the miniclone technique the replicative capacity of individual cells growing in bulk culture can be measured as well as the sizes of colonies generated by dividing cells (Ponton et al, 1983). Results showed that the percentage of glial cells capable of dividing gradually decreases with every new passage. This data is based on the broad distribution of colony sizes which showed a shift from many large colonies to more small colonies as population doublings increased.

Modern techniques for measuring the dynamics of normal cell populations involve measuring not only the senescent fraction of cells, but also the proliferating and apoptotic fraction. The most commonly used method to visualise senescent cells both in culture and in vivo is the senescence-associated beta-galactosidase assay (SA-β-Gal) (Dimri et al, 1995). Although this is a safer method than using 3H-thymidine labels, its robustness as a biomarker is questionable since the assay is dependent upon lysosomal mass (and cell size) rather than growth state (Lee et al, 2006). Cellular proliferation markers such as bromodeoxyuridine (BrdU) and Ki67 are commonly used to label and calculate the proliferating fraction. For measuring the apoptotic fraction, terminal transferase dUTP nick end labelling (TUNEL) is a commonly used method. This assay can detect DNA fragmentation that results from apoptotic signaling cascades.

An example of these methods being used for determining the growth dynamics of human umbilical vein endothelial cells (HUVEC) can be observed in a paper by Kalashnik et al (2000). Results show a gradual decline in the growth fraction as measured by Ki67, an increase in the senescent fraction and the apoptotic fraction remaining unchanged with each serial passage.

These findings thus suggest that the mechanisms resulting in cellular senescence is a stochastic process. As the proliferative capacity of cells declines with age or with increasing population doublings, the mechanism leading to cellular senescence is one in which these stochastic events gradually increases.