Cellular Senescence

Biochemical and Morphological Characteristics of Senescent Cells

To date, most studies describing the senescence phenotype have been performed in fibroblasts, although at least some senescence characteristics occur in epithelial and hematopoietic tissues as well. Despite induction of replicative and premature senescence by diverse stimuli, the biochemical and morphological characteristics of senescent cells are similar ( Figure 15-1 ). In vitro, senescent cells can be identified on the basis of their large flattened morphology, a lack of DNA replication (detected by the reduced incorporation of 5-bromodeoxyuridine or H-thymidine), increased expression of proteins associated with cell cycle arrest and tumor suppression (such as the tumor suppressor p53 and cyclin-dependent kinase inhibitors [CDKi] p16 ^INK4A , p21 ^CIP1/WAF1 , and p15 ^INK4B ), and the presence of senescence-associated β-galactosidase activity, which is attributed to the high lysosomal content of senescent cells. Senescent cells undergo marked changes in chromatin structure, as characterized by the presence of densely staining senescence-associated heterochromatic foci (SAHF), and also display altered histone modification profiles. Finally, senescent cells secrete a diverse array of proinflammatory and extracellular matrix remodeling factors, collectively referred to as the senescence-associated secretory phenotype (SASP).

The challenges associated with studying senescence in vivo have been a roadblock in the advancement of the senescence field. In contrast to apoptosis, another program that restricts proliferation and tumorigenesis, the morphological changes associated with senescence are difficult to visualize in whole tissues, and there are no simple assays (analogous to assessing DNA fragmentation or caspase activation during apoptosis) for definitively identifying senescent cells histologically. Thus senescent cells are predominantly identified in vivo by the presence of a collection of biochemical marks. Unfortunately, many “senescence markers” are not unique to the program; for example, upregulation of certain CDKi also occurs in quiescent cells. Moreover, the combination of biochemical marks expressed by given senescent cells can be cell-type and stimulus dependent. Despite these limitations, cells with combinations of senescent markers have been observed in aged, damaged and fibrotic tissues, in premalignant lesions, and in malignant tumors following chemotherapy, suggesting key processes in which the program might participate.

Replicative Senescence and the Hayflick Limit

Studies by Hayflick and colleagues demonstrated that most normal human diploid cells could not be subcultured beyond about 50 passages in vitro—the so-called Hayflick limit. On reaching this limit, cells remained irreversibly arrested with a senescent morphology, even when presented with growth factors in an optimal proliferative environment. Although later reports demonstrated that the cellular lifespan of clones within a bulk population is more variable than originally proposed, the original concept that normal cells invariably stop dividing in culture, even in optimal growth conditions, holds true.

Over the ensuing decades, the Hayflick limit was shown to occur as a consequence of accumulated telomere erosion and dysfunction. Telomeres are complex structures consisting of repetitive DNA sequences ([TTAGGG] _n in vertebrates) and specialized proteins that form protective caps on the ends of linear chromosomes to prevent their recognition as a DNA break. The “directional” nature of DNA replication prevents the replication of the extreme ends of telomeres (the “end replication problem”); thus telomeric DNA shortens with every cell division. With repeated divisions, telomeres can become critically short and fail to form the protective cap, resulting in activation of DNA damage signaling and the onset of replicative senescence and thereby preventing cellular immortalization. Even when senescence is prevented through a variety of genetic perturbations in the program, ongoing telomere dysfunction creates a state of chromosomal instability called crisis that limits proliferation.

In order for cancer cells to bypass senescence and become immortal, they must acquire an ability to regulate telomere length and/or integrity. The addition of telomeric DNA repeats can be catalyzed by the enzyme telomerase. On rare occasions cells can aberrantly upregulate the expression of telomerase (or elongate telomeres through alternative pathways), enabling bypass of replicative senescence and crisis. This effectively facilitates the unlimited propagation of cells with chromosomal fusions and genomic instability, a critical step preceding cellular transformation. Indeed, expression of telomerase alone is sufficient to delay or completely abrogate the onset of replicative senescence in certain cell types, which provides definitive evidence linking telomere shortening to the onset of senescence.

Most normal cells do not express telomerase and are therefore susceptible to replicative senescence. However, telomerase is expressed by normal cells that are dependent on long-term proliferative potential for their biological function, such as germ, stem, and progenitor cells. Although the expression of telomerase alone is insufficient to transform human cells, telomerase activity is often associated with human cell immortalization and is upregulated in many cancer cells. Moreover, expression of telomerase is a key factor in an oncogene “cocktail” capable of fully transforming normal human fibroblasts. Hence, strategies to target telomerase for cancer control have received much attention.