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CELLULAR AGING: TELOMERES

Aging is a complex process that occurs on multiple levels. The end result of aging is that life span is limited in multicellular organisms. The cells that make up multicellular organisms also have limited life spans. The limitation on cellular life span is comprised of two parts: (1) cells become unable to continue dividing but remain metabolically active, and (2) at some future time cell death occurs. Many cells in the human body are continually undergoing cellular division. Cellular division is a normal condition of certain tissues; examples include hair growth, the sloughing off of skin every several days, and the complete turnover and replacement of the cells of the immune systems every few months. In some instances, cellular division occurs in order to heal damaged tissues. Thus, having a limited number of cellular divisions available could contribute to aging by slowing down processes such as wound healing, as well as affecting general tissue maintenance.

In the 1960s, Leonard Hayflick first noted that human cells undergo a limited number of divisions when placed in culture. Furthermore, he noted that the number of divisions cells undergo is related to the number of prior divisions undergone by the cells. This observation suggested the existence of an intracellular clock that marked the division history of each cell. In addition, it suggested that once a predetermined number of divisions has occurred, a signal (or signals) is generated that prevents the cell from undergoing further divisions. The timing mechanisms underlying and regulating this process remained elusive until the end of the twentieth century. The first of these clocks to be identified and characterized, the telomere, is active in several human cell types.

Telomeres are chromosome caps

Telomeres are specialized structures present at the end of liner chromosomes; they serve the essential function of protecting and stabilizing chromosome ends. The telomere was first defined in the 1930s following observations that naturally occurring chromosome ends behave differently than chromosome breaks induced by damaging agents such as ionizing radiation. Both structures are ends of double-stranded DNA molecules. However, chromosome ends are stable, allowing accurate transmission of chromosomes from generation to generation without loss of genetic material, whereas induced breaks are very unstable, reacting with other chromosomes in the cell to create rearrangements and chromosome fusions. In addition, broken ends of DNA trigger cellular protective responses. These responses act either to allow the DNA damage to be repaired, or to remove the cell from the population by cellular suicide, called apoptosis. Even though telomeres are the physical end of a DNA molecule, they do not trigger these protective responses. These observations indicated that there is something special about naturally occurring chromosome ends.

Telomere structure

Telomeres are made up of short tandem repeats of a simple DNA sequence and associated proteins. In humans, and all other vertebrates, the telomeric DNA sequence is 5'(TTAGGG)3', oriented towards the end of one DNA strand, with the complimentary sequence 5'(CCCTAA)3' oriented towards the interior of the chromosome. The duplexed telomeric repeats are arranged in tandem and are present in more than a thousand copies at the end of each human chromosome. At the very end of the chromosome there is a single-stranded protrusion of the G-rich strand that extends for twenty or more repeats.

The first protein components of the human telomeric complex were identified in the mid-1990s. These proteins bind to the double-stranded telomeric repeats and are instrumental not only in promoting stability through formation of specialized structures, but also in regulating other aspects of the telomere, such as the number of repeats present. Within five years of identifying the first telomeric protein, TRF1, the number of proteins known to be present at human telomeres had expanded greatly. These included not only those that bound directly to the telomeric repeats, such as TRF1, and a related protein, TRF2, but also interacting proteins that serve to modify telomeric proteins, such as tankyrase, which binds to TRFI and adds to the protein long chains of ADP-ribose (a molecule which affects protein function). Finally, proteins that were previously identified as being involved in DNA repair and recombination have also been localized to telomeres, although the role of these proteins at the telomere remains unclear.

Experiments carried out by the laboratories of Titia de Lange and Jack Griffith in 1999 identified the something special that allows telomeres to impart stability on chromosome ends. These researchers purified telomeres and associated proteins from human and mouse cells and used electron microscopy to directly visualize the structure of mammalian telomeres. Their results demonstrated that the telomere exists as a dosed circular structure, called the telomere loop, or t-loop. The single-stranded DNA protrusion at chromosome ends, in combination with telomeric binding proteins, is critical in promoting the formation of the t-loop. This structure sequesters the naturally occurring ends of the DNA molecule, the telomere, rendering the chromosome end unreactive and invisible to the DNA damage-sensing machinery.

Telomeres and replication

In order for a cell to divide and create two equal daughter cells, the chromosomes must be replicated. Telomeres represent a unique challenge to the cell with respect to replication. Eukaryotic DNA replication occurs unidirectionally on each template strand. The enzymes that synthesize DNA, polymerases, require short RNA molecules to act as primers for new strand synthesis, and synthesis occurs in one direction only. Following extension, the primer is removed and the resulting gap filled in. This works well for most of the chromosome, but a problem arises at the very end of the chromosome. Upon removal of the final primer from the daughter strand, a gap remains that can not be replicated. This, in turn, would result in gradual loss of DNA from the chromosome ends each time the chromosome is replicated, and thus at each cell division. The inability of conventional cellular machinery to replicate the ends of DNA molecules came to be known as the end-replication problem. However, it was apparent that a mechanism for replicating chromosome ends existed, because chromosomes are faithfully transmitted to progeny.

Elizabeth Blackburn and Carol Greider first identified the enzyme responsible for telomere replication in a unicellular protozoan, Tetrahymena thermophila. This enzyme, called telomerase, is minimally composed of an RNA molecule and a protein subunit. The RNA molecule, in humans called hTERfor h uman te lomerase R NA, acts as a template to allow the addition of nucleotides to the end of the chromosome. The extension reaction is catalyzed by the protein component, in humans called hTERTfor h uman te lomerase r everse t ranscriptase. Thus, loss of telomeric DNA due to the end-replication problem may be balanced by an addition of telomeric repeats by telomerase. Telomerase is active in the germ line (the egg and sperm), where it acts to polish off the replication of chromosome ends so that each generation begins life with chromosomes similar to their parents. However, telomerase is not active in most cells of the body, with the result that DNA is gradually lost from the ends of our chromosomes each time a cell divides.

Telomeres and replicative senescence

As discussed above, cells have a finite division potential, often called the Hayflick limit. Interestingly, the number of divisions a cell is capable of undergoing in culture is inversely proportional to the age of the donor. That is, cells derived from younger individuals will undergo mote divisions than those from older individuals. Thus, the limitation on division potential is hypothesized to play a role in aspects of human aging. Cells that have reached their division limit undergo a process called replicative senescence, which is accompanied by morphological changes and changes in gene expression patterns. Interestingly, the Hayflick limit is ordained by the total number of divisions experienced by a cell and not by elapsed time. Senescent cells are alive (metabolically active), but can no longer be induced to divide. For many human cell types, the onset of replicative senescence has been linked to the length of the telomere.

The first clue that telomeres might play a role in capping the total number of divisions any given cell may undergo came from observations in the late 1980s made by Howard Cooke and coworkers. These investigators noted that telomeres in the germ line were longer than telomeres present in somatic tissue (i.e., blood) from the same individual. Over the next several years, a number of laboratories demonstrated that telomeres are shorter in older individuals than in younger individuals and that telomeres become shorter with increased numbers of cell divisions in culture. In addition, it was noted that chromosome instability of a type that would be predicted to accompany loss of telomere function, such as fusion of chromosome ends, is increased in older individuals. This was also observed in cultured cells as they approached senescence. There are two essential points to these observations. First, telomeres only shorten if cells divide. Metabolically active cells that are quiescent (those that do not divide) do not lose telomeric DNA. Secondly, telomeric DNA is only lost in somatic (body) cells, which do not, as a rule, contain telomerase activity. Based on these observations, it was proposed that the telomere might act as the elusive intracellular clock that triggered senescence. This became known as the telomere hypothesis of cellular aging. This hypothesis suggests that attrition of telomeric DNA eventually compromises telomere function. This would result in a signal being generated that causes the cell to undergo replicative senescence and cease dividing. Although these observations were suggestive of a causal relationship between functioning telomeres and the ability to divide, the link between telomere loss and replicative senescence remained correlative.

Following the identification and cloning of the RNA and catalytic components of telomerase, it became possible to force expression of this enzyme in primary human cell cultures. Primary human cell cultures have a finite division capacity and do not contain telomerase activity. In the late 1990s it was finally directly demonstrated that, for specific cell types, expression of telomerase and the concomitant extension of telomeric DNA is sufficient to impart cellular immortalitythe potential for an infinite number of divisions. The inverse is true as well. Thus, inhibition of telomerase activity in immortal cells, such as tumor-derived cell lines, results in telomere loss and culture senescence. These experiments directly demonstrated a causal link between maintenance of telomeric DNA and a cells ability to divide. In a series of experiments, de Lange and coworkers demonstrated that disruption of telomeric structure by removing the telomeric protein TRF2 resulted in loss of protective function and a senescent-like growth arrest. These experiments identified the first type of signal that might emanate from telomeres to elicit cellular responses. According to the t-loop model described above, loss of TRF2 would open the end of the chromosome by disrupting the t-loop. This, in turn, alarms the cell because the telomere now resembles a broken DNA molecule and results in activation of the ATM-dependent and p53-dependent DNA damage response pathway. Activation of p53 has been linked to both senescence and apoptosis. Thus, cells that are unable to sequester chromosome ends through maintenance of the t-loop structure, either because the telomere is too short or due to absence of essential proteins, are prevented from dividing further by activating the senescence or apoptotic pathways. The question of whether complete loss of function is required to evoke senescence, or whether the cell has some means of identifying a short but still functional telomere, has yet to be answered.

Telomeres and premature aging syndromes

There are several human syndromes that manifest as premature aging, including Hutchinson-Gilford progeria and Werner syndrome. Cell lines derived from individuals with progeria are capable of attaining fewer divisions before becoming senescent than are cells from age-matched unaffected individuals. In addition, telomeres in cells derived from individuals with Hutchinson-Gilford progeria are shorter than age-matched unaffected individuals, again linking telomere length with cell division capacity and, indirectly, with aging. Interestingly, experiments have demonstrated that cells derived from individuals with Werner syndrome not only undergo senescence after fewer divisions, but also that this occurs when the telomeres in these cells are, on average, longer than those in parallel cultures not containing the Werner mutation. This observation suggested that the premature senescence in cells derived from individuals with Werner syndrome might be disconnected from telomere length, and instead result from other factors, such as accumulated DNA damage. However, David Kipling and coworkers demonstrated that forced expression of telomerase in Werner syndrome cells conferred immortality, indicating that the telomere-length-based clock is active in this genetic background.

The link between telomere shortening, replicative senescence, and aging at the organism level is supported by a series of studies carried out by the laboratories of Ronald DePinho and Carol Greider. These investigators generated a mouse that was deficient for telomerase. Mice usually have extremely long telomeres, but in the telomerase-deficient mice the telomeres became shorter with each generation, and the later generations of these mice exhibited characteristics consistent with premature aging. For example, these animals have a compromised ability to heal wounds, exhibit premature graying, and have a shortened lifespan. These studies linked short telomeres with some characteristics observed in older adults, suggesting that telomere-length-dependent effects on cellular division might also play a role in aging at the level of the organism. The strength of the contribution of telomere-length-based replicative senescence to the aging process as a whole remains to be determined, however.

Telomeres as tumor suppressors

The observations discussed above clearly linked telomeres to the ability of cells to divide and proliferate. Simultaneously with these studies, and key to the development of the telomere hypothesis of cellular aging, the link between telomere stabilization and tumorigenesis was becoming clear. One feature of tumor cells is their unlimited cell-division potential, or immortality. It was first reported in 1990 that telomeres were shorter in tumors than in adjacent healthy tissue from the same individual. These observations prompted the suggestion that the cell divisions leading to tumor formation resulted in telomere loss. However, telomere length in tumor-derived cell lines remain stable over time in culture, despite continuing cell division. This observation indicated that cells that have transformed and become tumors have some means of overcoming the end-replication problem. The obvious candidate for achieving stabilization of telomeric DNA was telomerase. Studies in the early 1990s did, in fact, suggest that this enzyme was active in some tumors and immortal cell Lines. However, these studies were hampered by the lack of sensitivity of the assay used to detect telomerase.

A highly sensitive assay to detect telomerase activity was developed in 1994, and shortly thereafter, surveys of cell lines and tumors were begun to determine if telomerase activity was a common feature. The unambiguous results were that the majority (8090 percent) of all human tumors, as well as the majority of immortal transformed cell lines, contain telomerase activity. One implication is that telomerase activation would stabilize telomere length and allow the cell to circumvent cellular senescence. However, the timing of telomerase activation during tumori-genesis is unclear, and some discussions suggest that this may occur after at least a transient abolition of telomere function (see below). These observations, together with the links between telomeres and cellular senescence, have provoked the suggestion that telomere length might act as a tumor suppressor mechanism by limiting the number of divisions any given cell might undergo.

Telomeres, genome stability, and cancer

The hallmark of telomeres is their ability to confer stability on chromosome ends and prevent chromosome ends from activating the cellular surveillance mechanisms protecting cells from the deleterious effects of DNA breaks. Chromosomes with critically short telomeres become compromised in chromosome-end stability, and, because telomeres are generally shorter in tumors than in adjacent healthy tissue, it was suggested that telomere dysfunction could contribute to tumorigenesis by increasing genomic instability. In the short term, this genomic instability might promote tumor formation by allowing growth-advantageous mutations (i.e., mutations that permit cells to remain viable under conditions when, normally, the cells would die) to accumulate rapidly. However, this scenario requires that the cellular mechanism(s) that normally monitor telomere length and prevent cells with critically short telomeres from dividing be blocked. Under these conditions, cells with critically short telomeres would continue dividing. These cells would enter a period of extreme chromosomal instability. Eventually, however, such rampant genome instability might prove deleterious to tumor survival by generating mutations in genes that are essential for cellular survival. Thus, stabilization of the genome, perhaps through activation of telomerase, would have a selective advantage. Experiments carried out by the DePinho and Greider groups utilizing telomerase-deficient mice support the idea that transient telomere malfunction may promote tumorigenesis.

Telomeres, aging, and cancer

The experiments described here link telomeres to two sides of a single coin: cellular mortality and immortalityor aging and cancer. The salient points with respect to cellular aging, given the assumption that replicative senescence as observed in cultured cells contributes at some level to aging, are: (1) telomeres are shorter in somatic (body) cells than in the germ line (egg and sperm), (2) telomeres are shorter in older individuals than in younger individuals, and (3) telomeres get shorter with increased number of cellular divisions undergone in culture. On the flip side of the coin, most tumors have bypassed the telomere-length-dependent limitation on cellular proliferation, usually through the activation of telomerase. Finally, stabilization of telomere length by forced expression of telomerase is sufficient to confer an unlimited division potential, or cellular immorality, while inhibition of telomerase with concomitant telomere shortening causes growth arrest in immortal cells.

There is, however, an important caveat to the arguments discussed here. As noted above, telomere length acts as a type of clock in many human cell types. However, there are cell types that utilize some other clock mechanism to limit the number of divisions they undergo, including thyroid epithelial cells and breast epithelial cells. These cells enter replicative senescence while still having telomeres of sufficient length for many more divisions. Furthermore, simple expression of telomerase is not sufficient to confer immortality on these cell types. It is believed that even these cells, given circumvention of their primary clock, eventually become dependent on telomere length for continued division. The observation that most human tumors, including thyroid and breast tumors, have telomerase activity and stable telomeres would support this idea. Thus, the cumulative data indicate a key role for telomeres in the processes of cellular senescence and tumorigenesis.

Dominique Broccoli

See also Cancer Biology; DNA Damage and Repair; Genetics: Longevity Assurance; Theories of Biological Aging.

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Cellular Aging: Telomeres

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