Most eukaryotic cells undergo a finite number of cell divisions. Cell division is restricted by a process of replicative or cellular senescence where cells reach an irreversible arrest of cell proliferation. Several genes are known to play a role in the control of replicative senescence. There are also several hypotheses to explain the aging process in eukaryotic cells. The characteristics of aging are unique and affect all individuals and are separate from diseases that result due to the aging process.
Normal eukaryotic cells do not divide indefinitely. The replicative span of cells is limited and is referred to as replicative senescence. The finiteness of human cells was first described by Hayflick in the 1960s using human fibroblasts. Replicative senescence depends on the number of cell divisions—not the time taken for cells to complete the cell cycle. It is also dependent on the cell type. While some cells such as the germ line cells divide indefinitely, most other normal cells retain the capacity to senesce with age.
Proliferating cells reach their limit largely because repeated DNA replication in the absence of the enzyme telomerase (Chapter 7) causes telomeres to progressively shorten. Cells that fail to senesce proliferate despite dysfunctional telomeres. Eventually these cells develop aberrations in their chromosomes that can result in cancer. Therefore, the senescence response represents a fail-safe mechanism in place to prevent malignant transformation. Accumulation of DNA damage, inappropriate expression of oncogenes, and generation of reactive oxygen species (ROS) are all known to induce senescence (Figure 24.1).
FIGURE 24.1.Senescence inducers.
The senescent phenotype
Senescent cells display a number of phenotypes that distinguish them from quiescent cells, and in this manner, senescent cells are thought to enter a form of terminal differentiation state. This state cannot be reversed by physiological stimulators of growth. Senescent cells, while incapable of replication, are generally viable and metabolically active, survive long term, and resist apoptosis (Figure 24.2). These cells also express a senescence-associated beta-galactosidase enzyme activity (SA-B-gal).
FIGURE 24.2.The senescent phenotype.
1. Irreversible cell cycle arrest: Growth arrest of senescent cells occurs mostly in the G1 phase and is accompanied by an increased expression of cell cycle inhibitors and a decreased expression of cell cycle regulators such as cyclins and transcription factors (E2F) (see Chapter 20) necessary for progression of the cell cycle.
The cyclin-dependent kinase inhibitors p21 and p16 are important players in aging and age-related disease. They function to inactivate the cyclin-dependent kinases (Chapter 20) which maintain the retinoblastoma protein (pRB) in its active hypophosphorylated state necessary to produce a block in cell cycle progression in G1.
2. Chromatin modification during senescence: There is age-associated remodeling of chromatin. While there is a genome wide decrease in methylation, there is also an increase in methylation at specific sites in the genome. This happens at CG sites (Chapter 6) on DNA, which may comprise of promoter regions of genes. The decrease in methylation happens in regions comprising of repetitive DNA sequences. Histones (Chapter 6) are also modified with age by deacetylation, creating unique regions in the chromatin called senescence-associated heterochromatin foci (SAHF). This type of modification is usually seen when proliferation is triggered in senescent cells, causing regions associated with proliferation-promoting genes to be silenced and contributing to the senescence-associated proliferation arrest (Figure 24.3).
FIGURE 24.3.Senescence-associated proliferation arrest.
One class of proteins that play a role in the protection of cell from age-related decline in function is sirtuins. Sirtuins (founding member—silent information regulator 2) are NAD-dependent histone deacetylases that are evolutionarily conserved. The uniqueness of sirtuins lies in the fact that unlike deacetylases, which simply hydrolyze acetyl-lysine residues, the sirtuin-mediated deacetylation reaction couples lysine deacetylation to NAD hydrolysis.
This hydrolysis yields, in addition to other products, nicotinamide, which itself is an inhibitor of sirtuin activity. The dependence of sirtuins on NAD links their enzymatic activity directly to the energy status of the cell via the cellular NAD:NADH ratios. While other enzyme activities are known for this family of proteins, sirtuin 1 (SIRT1) (human counterpart of sirt2) is an NAD-dependent histone deacetylase with roles in chromatin silencing and deacetylation of lysine amino acids in other nonhistone target proteins.
It has been found to be the key molecule that affects longevity within the context of calorie restriction (see clinical correlation), which has long been known to increase life span.
Progerias and nucleus architecture
Hutchinson-Gilford progeria syndrome (HGPS) is a rare syndrome that is characterized by greatly accelerated aging. These individuals demonstrate severe growth retardation, loss of subcutaneous fat, reduced bone mineral density, alopecia, and poor muscle development. They also show wrinkled skin and have a higher incidence of stroke and myocardial infarction. The average age of death for individuals affected by this syndrome is 12 to 15 years.
Interestingly, these patients do not present with all aspects of aging. In fact, they do not show increased incidence of cancers, neurodegeneration, and other age-related disorders such as arthritis or cataracts. At the molecular level, HGPS may represent accelerated aging. The syndrome is caused by a mutation in the Lamin A gene which encodes a nuclear scaffold protein. The nuclei of HGPS cells show dramatic changes in structure (Figure 24.4) and the changes to chromatin are similar to that seen in the normal aging process.
FIGURE 24.4.Hutchinson-Gilford progeria syndrome (HGPS) and nuclear architecture.
Mechanisms involved in senescence induction
Many mechanisms have been proposed to contribute to the aging phenomenon in eukaryotes; however, the specific contribution of each and their relative importance in this process is a matter of debate.
1. DNA replication stress: This is defined as inefficient DNA replication that causes DNA replication forks to progress slowly or stall. Many factors are known to contribute to this type of stress to DNA replication (Figure 24.5). RecQ helicases are a group of helicase enyzmes that are important in maintaining the genome. They function in unwinding DNA and aiding in replication of double-stranded DNA. In the absence of these enzymes, replication does not proceed normally and is implicated in the process of aging. Mutations in the WRN RecQ helicase cause genomic instability and the premature aging syndrome Werner syndrome. In addition to shortened life span, Werner syndrome is characterized by premature graying and thinning of hair, osteoporosis, type II diabetes, cataracts, and an increased incidence of cancer.
FIGURE 24.5.DNA replication stress.
2. Mitochondria, reactive oxygen species, and aging: Mitochondria have their own genome and they replicate and transcribe their DNA semiautonomously. Like nuclear DNA, mitochondrial DNA (mtDNA) is constantly exposed to DNA damaging agents. The free radical theory of aging proposes that aging as well as the associated degenerative diseases could be attributed to the deleterious effects of free radicals on various cellular components. One of the sources for ROS in the cell is oxidative phosphorylation within mitochondria, so the free-radical theory of aging is essentially a mitochondrial theory of aging. Because mtDNA is vulnerable to oxidative stress (due to the close proximity between the sites of ROS production and mtDNA), mtDNA mutations accumulate progressively during life and are directly responsible for a measurable deficiency in cellular oxidative phosphorylation activity, leading to an enhanced ROS production. The increase in mutational load in mtDNA combined with increased ROS production creates a “vicious cycle” that culminates in the aging of the organism (Figure 24.6).
FIGURE 24.6.Mitochondria, reactive oxygen species, and aging.
Calorie restriction, aging and red wine
Caloric restriction, which is the reduction of caloric intake without malnutrition, is known to be important for extending the life span in a wide range of species including humans and for postponing the manifestations of aging, including both functional decline and age-related diseases. Much is known about the physiological changes that occur when subjected to caloric restriction, but molecular mechanisms are not known.
Recently, sirtuins have been found to be induced by calorie reduction. Resveratrol, a polyphenolic compound in red wine, which has been shown to mimic calorie restriction in its ability, potently induces SIRT1 expression in addition to a number of related protective effects on cell metabolism aimed to prevent age-related decline in physiological functions. The amount of resveratrol necessary is the caveat—requiring an equivalent of 100 bottles of red wine to produce the beneficial effect seen in laboratory animals!
3. Stem cell theory of aging: According to this theory, tissue-specific adult stem cells, which are important for self-renewal and tissue replacement during the human life span, show an age-associated decline in replicative function. These cells are generally retained in the quiescent state but can be induced into the cell cycle in response to physiological growth factor stimulation even after prolonged periods of dormancy. Once stimulated, the stem cells can produce undifferentiated progeny of cells, which, in turn, produces differentiated cells through subsequent rounds of proliferation. It is thought that aging affects the ability of stem cells to produce the undifferentiated progeny and the differentiated cells but not its ability to self-renew (Figure 24.7). According to this model, one of the mechanisms used by tumor suppressors such as p53 is in part through activation of differentiation in the self-renewing tissue-specific stem cells. P16 has also been shown to play an important role in the age-related decline of stem cells. Sufficient evidence is available to indicate that an age-associated increase in p16 levels restricts self-renewal and upsets tissue homeostasis (see below).
FIGURE 24.7.Stem cell’s functionality is altered with age.
Molecular mechanisms in senescence response
While diverse stimuli can induce the senescence response, they all appear to converge on either one or two different pathways that both establish and maintain the senescence response. These pathways are governed by two tumor suppressor proteins, p53 and pRB (Chapter 21).
1. The p53 pathway: It is well known that p53 is an important mediator of cellular responses to DNA damage. It has also been found to be an important mediator of senescence response as attrition of telomeres (which resembles damaged DNA) similarly triggers an increase in p53. Inappropriate activation of oncogenes within normal cells also induces a senescence response through activation of p53. Oncogenes such as RAS (Chapters 17, 22) are known to signal through generation of ROS, which is necessary for their mitogenic effects. However generation of DNA damaging ROS also activates the p53-dependent damage response. At least, in some cell types, the induction of senescence by DNA damage, telomere dysfunction, and possibly oncogene overexpression converge on the p53 pathway, which is both necessary and sufficient to establish and maintain senescence arrest. Although the senescence-induced growth arrest cannot be reversed by physiological growth signals, it can be reversed by inactivation of p53 function, which explains the age-related increase in cancer incidence in the population (Figure 24.8)
FIGURE 24.8.Molecular mechanisms in senescence response.
2. The pRB pathway: In some cells, p53 inactivation alone appears to be insufficient to reverse the senescent phenotype. The difference appears to be in the presence or absence of p16 expression. Stress increases the expression of p16 and the resultant increase in pRB is known to aid in the reorganization of the chromatin, resulting in replicative senescence-related inhibition of gene encoding cell cycle regulators.
- Most eukaryotic cells undergo a finite number of cell divisions.
- Replicative senescence refers to the number of cell divisions that a cell undergoes before it irreversibly ceases proliferating.
- Senescent cells are metabolically viable but will not enter the cell cycle under any condition.
- Senescent cells are characterized by increased expression of cell cycle inhibitors such as p16 and p21.
- The chromatin in senescent cells demonstrates a unique structure as a result of modifications to histones and DNA.
- Sirtuins are a group of proteins with NAD-dependent deacetylase activity that offer protection against age-associated decline in metabolism and physiological functions.
- Mitochondria-associated ROS formation is implicated in the oxidative damage to macromolecules coupled with a decrease in oxidative phosphorylation.
- Age-associated decline in the replicative potential of stem cells also accounts for the deficiencies in tissue replacement during aging