In the 1930s, Barbara McClintock, who studied corn, and Herman Muller, who studied fruit flies, collaboratively theorized that chromosomes have specialized ends to prevent fusion. This theory was based on a phenomenon observed by McClintock: X-ray treatment of corn caused the formation of circular ring chromosomes. The chromosomes that circularized had lost their protective ends, which she named telomeres (1). McClintock and Muller had unwittingly uncovered Hayflick's "internal time clock" which is described in the Immortalization section. In 1978, Elizabeth Blackburn found that telomeres in Tetrahymena had hundreds of base pair repeats of the sequence TTGGGG. In 1985, Carol Greider discovered telomerase, the enzyme that synthesized the telomeric repeats in Tetrahymena. Finally, in 1988, Moyzis lab sequenced the human telomere and found the 6 base pair repeat TTAGGG (1). Elizabeth Blackburn, Carol Greider and Jack Szostak were awarded the 2009 Nobel Prize in Medicine and Physiology for their research leading to the discovery of telomeres and the enzyme telomerase.
The End Replication Problem
Telomeres are located at the ends of linear chromosomes and are composed of non-coding DNA consisting of multiple base pair repeats and associated protein factors. In humans, these TTAGGG repeat sequences are typically 8-14 kbp long (2). In 1972 and 1973, respectively, Watson and Olovnikov discovered the end replication problem that causes telomere shortening. Chromosomes lose some telomeric DNA after each cell cycle phase due to limitations of the DNA machinery. The polymerase replicates only in the 5' to 3' direction, so there are variations in replication between the leading and lagging strands of DNA. The leading strand is continually being replicated. On the other hand, in lagging strands, Okazaki fragments and RNA primers are necessary for the the polymerase to start replication. These primers get degraded and are subsequently replaced by DNA. However, this causes a gap on the last Okazaki fragment resulting in the so-called end replication problem. This predicts a progressive reduction of chromosomal DNA at its 3' ends as the cell moves through multiple cell cycles. Each round of DNA replication leaves 50-200bp of un-replicated DNA at the 3' end. Cellular senescence is triggered when telomeres become on average 4-6kb long (3). Telomeres exist as a solution to the DNA end-replication problem. While telomeres do not prevent the physical shortening of chromosomes, they do act as disposable DNA, which can be sacrificed to protect the integrity of the important coding regions contained within the chromosome. Without telomeres, the chromosomal ends would slowly be eroded with each round of DNA replication (4). This erosion is seen throughout the human lifespan, as at birth, the length of a human telomere is anywhere between 15 and 20 kilobases long (5) and with each cell division they shorten by 50-100 base pairs (6).
Telomere shortning can also be aggrandized by numerous factors. Epel et al. (2004) evidenced accelerated telomere shortening with psychological stress, finding that women with the most stress displayed telomeres shorter by the equivalency of a decade compared to women with low stress (12). Likewise, von Zglinicki (2002) demonstrated that telomere shortening is attributed to increased oxidative stress (13).
Telomere Structure and the Shelterin Complex
In vertebrates, telomeres are comprised of the DNA tandem repeat sequence TTAGGG, the length of which shows extensive variation between species (8). Mammalian telomeres have a 150-200 bp G-rich 3’ overhang which embeds itself into the upstream telomeric region, forming a T-loop structure. The DNA that is displaced by this process forms what is known as a D-loop (2). This structure creates a terminal cap that protects against degradation and double strand break repair machinery, preventing recombination and non-homologous chromosome joining (2). This region also regulates telomere elongation by telomerase (2).
A number of proteins associate with telomeres, forming the shelterin complex (1). The proteins TRF1, TRF2, and POT1 bind specifically to telomeric DNA. POT1 is unique in that it binds single-stranded DNA. TIN2 forms a complex with TRF1/TRF2. TPP1, responsible for recruiting the telomerase holoenzyme to the end of the chromosome, binds to TIN2 and POT1 (2). RAP1, the final component of the shelterin complex, is not necessary for telomere capping, but instead impedes telomere recombination when bound to TRF2. Together, the proteins of the shelterin complex act to stabilize and promote elongation of the telomere.
Function and Maintenance
Telomeres function to protect chromosomal ends from enzymatic degradation. The ends of chromosomes without proper telomeres tend to fuse, and thus these chromosomes are lost during the cell cycle. Additionally, telomeres confer chromsomal stability and aid in setting up the nucleus by providing attachment sites for the nuclear matrix (2). The events of crisis are triggered when cells lose functional telomeres from their chromosomes. This is because the cell recognizes the shortened telomeres as broken or damaged DNA. If DNA is damaged, the cell either activates DNA repair enzymes or dies through apoptosis (3).
Normal cells do not express telomerase, the enzyme that is necessary for the regeneration of telomeres, so the telomeres continue to shorten from each cell division. The cell will send out damage signals when the telomeres reach a certain length, telling the cell to undergo telomere-dependent senescence. Upon detection of a short telomere, the cell will activate its DNA damage repair machinery and up-regulate p53, an important tumor suppressor involved in cell cycle control and tumour suppression. In turn, p53 will up-regulate p21, a kinase which inhibits cell cycle promoting cyclin proteins, resulting in the permanent arrest of the cell cycle at the G1 checkpoint (7). When p53 is inactivated, the cells will continue to divide when they should be undergoing senescence. Because the telomeres in p53 (-) cells continue to shorten, chromosomes become unstable and the cell undergoes crisis (2). Since telomere length determines how many times the cell can divide, it controls mitotic capacity. Cells able to replenish their telomeres can divide infinitely (immortalize). Immortalized cells tend to have high numbers of telomeric repeats, which are typically generated by active telomerase. Human tumors, unlike most normal human cells, can express telomerase. Other types of tumor cells that do not express telomerase can stabilize their telomere length by the alternative lengthening of telomeres (ALT) pathway. The ALT pathway is described in the Alternative Lengthening of Telomeres (ALT) Pathway section. Telomerase is a very important enzyme in cancer cell proliferation; its structure and mechanism of action will be discussed in the next section.
Cells keep dividing until they reach the end of their replicative lifespan, as determined by the Hayflick limit. Once telomeres shorten to a critical point, cells are unable to divide and undergo either senescence or crisis (1). Some cells are able to overcome the senescence associated with cell cycle arrest and therefore are able to continue to divide past their normal life cycle. These cells are not yet immortal and are unable to divide indefinitely. After an additional 20-30 cell divisions, the cells enter a state known as crisis (3). During this phase, the cells still divide, but undergo high levels of apoptosis (cell death) since high levels of chromosomal abnormalities exist at this time. There is, however, no overall increase in cell number. The 1 in 10^7 cells that do emerge from the crisis state are considered to have acquired immortality, and are able to divide indefinitely (3). As mentioned in the previous section, senescence represents a stop in cell division with retention of cell viability over extended periods of time, while crisis involves death of cell by apoptosis. Senescent cells have reasonably stable chromosomes while cells in crisis have widespread chromosome instability.
Detection of Telomere Length
As discussed above, the length of the telomeres is crucial for cell survival, proliferation, and senescence. It is therefore of interest to study the length of the telomeres of various cells at various stages. Currently, there exist few methods for determining telomere length, each with its strengths are caveats. Below are two of the commonly used techniques. (9)
Terminal Restriction Fragment (TRF) Analysis
TRF analysis was the first method used to measure telomere lengths, and for a long time, was the only method. Because TRF analysis measures the precise length of the telomeres in kb, it is now considered the “golden standard” of which all other techniques reference to. TRF is based on analysis of Southern blotting of the DNA telomeres of interest. On the downside, the presence of subtelomeric DNA in the TRF assay prevents this assay from being a suitable for establishing mean telomere lengths of cell unless multiple TRF assays are performed. In addition, TRF analysis is labour intensive, costly, and requires a greater amount of initial DNA. Determination of the physical telomere length is accomplished by comparing chemiluminescence telomere complementary probes bound to digested DNA Southern blot to a DNA ladder as well as internal DNA reference. By measuring the distance traveled of the sample, compared and referenced to the DNA ladder, the precise length of the telomere can be determined. (10)
Whereas TRF analysis measures precise telomere length, methods exist to study mean length as well. One of the more popular methods is based on qPCR with the protocol designed by Richard Cawthon. By measuring telomere length with qPCR, this method is low-cost and high-throughput, requiring very little initial DNA when compared to TRF analysis. The disadvantages of qPCR-based techniques largely center on the limitation of measuring mean rather than individual telomere length, as well as a high coefficient of variation (>2%). The principle of Cawthon’s technique is to compare telomeres to a single copy gene 36B4. By using different primers (T and S primers), T primers bind to telomeric repeat sequences, while S primers bind to the single copy gene 36B4. The ratio of the amplified telomere repeat copies to the number of single copy gene (T/S ratio) corresponds to the relative length of the telomere within the sample. It is important to run a standard curve of known DNA concentrations to compare the sample T/S ratios as a means of determining mean telomere length. (11)