Cellular Aging: Basic Phenomena

The failure to confirm the relationship between donor age and proliferative life span does not invalidate cell culture as a model for aging studies, because other significant alterations occur during senescence in vitro that also occur during aging in vivo. These changes include an increased number of copies of genetic material, increased cell size, decreased mitogenic response to growth signals, a decline in the expression of genes involved in growth control, increased expression of genes of the extracellular matrix, and various changes in cell morphology. In addition, a correlation between replicative capacity and initial telomere length of cells derived from donors up to ninety-three years of age has been reported.

However, not all changes that occur during senescence in vitro occur during the aging of organisms. For example, the induction of the c-fos gene following stimulation with serum is lost in senescent cells but is undiminished in old individuals. Even markers that have gained relatively widespread use have been found to detect different phenomena when they occur both in vivo and in vitro. One marker thought to be potentially universal was reported by Dimri et al. (1995), who observed increases in cytochemically detectable β-galactosidase activity (SA β-gal) at pH 6.0—both in cell cultures and in tissue sections obtained from old donors. They also observed that immortal cells exhibited no SA β-gal staining under identical culture conditions. Additionally, they interpreted their results as providing a link between replicative senescence and aging in vivo. These observations appear to be supported by studies in rhesus monkeys. This model has gained widespread use as a crude measure of senescence; however, a number of subsequent studies have shown that it is actually not as specific or universal as first claimed. For example, SA β-gal positive cells have been observed in quiescent cultures of mouse cells as well as some types of human cancer cells that were chemically stimulated to differentiate, even though none these cell types can be classified as senescent. Furthermore, one biochemical analysis demonstrated that β-gal activity was present at both pH 6.0 and pH 4.5 in a number of different tumor cells. Other studies failed to detect any correlation with donor age either in tissue sections or in skin fibroblast cultures established from donors of different ages. It was also observed that staining in skin sections occurred only in the lumen of sebaceous glands and shafts of hair follicles, which is consistent with detection of microbial invasion, rather than actual changes in the biochemistry of the human cells in tissue sections (Severino, et al., 2000). The source of these discrepancies cannot be entirely elucidated; however, it is clear that this type of staining cannot be used as a marker for all types of aging under all conditions.

An important biomarker that is linked to the cessation of mitotic activity associated with cellular senescence is the progressive loss of chromosomal telomeric repeats. The telomeres of human chromosomes are composed of several kilobases of simple repeats: (TTAGGG)n. Telomeres protect chromosomes from degradation, rearrangements, end-to-end fusions, and chromosome loss. During replication, DNA polymerases require an RNA primer for initiation. The terminal RNA primer required for DNA replication cannot be replaced with DNA, which results in a loss of telomeric sequences with each mitotic cycle of normal cells. The observation that telomere shortening correlates with senescence provides an attractive model for the way in which cells might "count" divisions. Telomere shortening has been observed in aging models both in vitro and in vivo.

An examination of some biomarkers of senescence lends at least some support to the hypothesis that the phenomenon occurs in vivo. For example, the messenger RNA (mRNA) for the angiogenesis inhibitor EPC-1 declines more than one-hundred-fold with proliferative aging in culture. Although the EPC-1 mRNA is readily detected in tissue sections from both young and old donors, the total amount of EPC-1 mRNA is also lower in older individuals. Furthermore, it is present in a mosaic pattern in the skin sections from older individuals. Thus, the decrease occurs only in some cells. Similarly, the average length of telomeres, which decreases progressively with increasing numbers of mitoses, exhibits large variations in multiple subclones established from one individual, again suggesting different telomere lengths are associated with different tissue regions in vivo. These observations suggest that loss of proliferative potential, in vivo, occurs in a mosaic pattern, and that both long- and short-lived cells may lie in close proximity in tissues. In a culture environment, the cells with the greatest growth capacity will ultimately become predominant in the culture. Hence, the selection during the procedure to establish cultures probably obscures differences in proliferative capacity that exist in vivo.