Even though the role of telomeres in protecting the end of chromosomes had been acknowledged for a long time, the mammalian telomeric sequence has only been identified more recently.[5, 16]It is now known to consist of tandem repeats of TTAGGG, and be 10 – 15 kilo-base pair (kbp) in length.[17] Telomeres effectively act as a biological clock, which allows human foetal cells to divide only 40 – 70 times during a typical lifespan.[18, 19] Telomeric DNA regions are bound to the various shelterin protein molecules to form a stable complex that inhibits DNA repair systems from acting on the telomere. The shelterin proteins include the Protection of Telomeres Protein 1 (POT1) and Telomeric Repeat-binding Factor 1 (TRF1).
4 Together with the other telomeric proteins, these molecules protect chromosomes from deterioration and telomere-telomere fusion events, by forming an unusual T- loop configuration shown in Figure 1.2 a.[20] The T-loop is formed by using the telomeric single strand overhang, which contains 150 – 200 nucleobases, to invade the telomeric double stranded region, and create a displacement loop, or D-loop, at the invasion site.[6] Before DNA replication occurs, this T-loop structure is opened and the ssDNA overhang at the 3´-terminal becomes accessible for extension by telomerase (Figure 1.2 b).[20]
Figure 1.2 Telomere structure:(a) T-loop configuration showing different telomeric length regulation proteins, including Telomeric Repeat-binding Factor 2 (TRF2), Repressor- Activator Protein 1 (RAP1), TRF1-interacting nuclear protein 2 (TIN2) and Tripeptidyl- Peptidase 1 (TPP1); and (b) Stretched configuration observed during telomere elongation process, showing some of the components of telomerase including Telomerase Reverse Transcriptase (TERT) and Telomerase RNA (TERC). Adapted from various references.[20- 22]
(TTAGGG)n (AATCCC)n 5´
3´
T-loop
D-loop
TERT-TERC complex
5´
G G G A T T G G G A T T G G G A T T G G G A T
T TTAGGGTTAGGGTTAGGGTTAGGG
AATCCCAATCCCAATCCCAATCCCAATCCCAATCCCA
RAP1
TIN2
POT1 RAP1
TIN2
POT 1
3´
Over hang ssDNA Telomeric dsDNA
region
TRF1
TRF2
RAP1 TIN2
POT1 POT1 POT1
POT1 TRF1 TRF2
RAP1 TIN2 TRF1 TRF2
TIN2
RAP1 TRF1 TRF2 RAP1
A AUCC C
5´
3´
5´
3´
TRF1 TRF2
RAP1
TIN2
POT1
TRF1
TRF2 TRF1
TRF2
TIN2
(a)
(b)
5 Telomerase elongates telomeric DNA sequences,[19] and was discovered in 1985.[23] It is a protein complex consisting of two core components. These are a catalytic subunit called Telomerase Reverse Transcriptase (TERT), and a Telomerase RNA component (TERC) which is a non-coding RNA sequence (AAUCCC in mammals) that serves as a template for telomere replication.[20]
Recently, cryo-electron microscopy studies have shown that the structure of telomerase is comprised of two flexible tethered lobes. In one lobe, the RNA wraps around TERT to form an organised tertiary structure for the catalytic core whereas the other lobe is an H/ACA ribonucleoprotein.[24] Purification and crystallization of telomerase has proved to be extremely challenging owing to its insolubility and low abundance.[25] Telomerase is highly upregulated in embryonic stem cells in order to help preserve genomic stability during a large number of cell division cycles.[20] It has also been found in other cells which divide regularly such as sperm cells,[6]
epidermal cells and bone marrow.[26] In contrast, telomerase is inactive in most somatic cells, which form the majority of tissues in the human body.[6] Intriguingly, Wright and co-workers showed that transfecting human cells with a vector encoding the hTERT protein allowed the cells to surpass their normal lifespan by at least 20 more cell division cycles.[18, 27] In addition, a correlation between overexpression of telomerase and uncontrolled cellular growth has been observed in numerous types of cancer cells. For example, it was reported in 1994 that telomerase was over- expressed in over 85% of cancer cell types, but in only a small proportion of specialized, normal healthy cells.[28] These observations together suggest that inhibition of telomerase might be a promising approach for the development of new cancer therapeutics.
6 It is a somewhat contradictory observation that normal cells with significantly low levels or a complete absence of telomerase have telomeres that are much longer than those in cancer cells which generally have significantly higher levels of the enzyme. This discrepancy, however, may be explained by examining the pathway by which malignant tumors are formed from normal cells (Figure 1.3).[19]
Depending on the living habits of different individuals, and the rate of mitosis in the cell cycle, the length of telomeres in normal cells is gradually shortened by approximately 15 – 28 base pairs per year. This continues until the telomeric DNA reaches the critical length of 4 – 6 kbp. At this point, the cells enter senescence which is called mortality stage 1 (M1) or the Hayflick limit.[5, 17, 29] The shortened and therefore defective telomeres trigger a DNA damage response (DDR) by
activating number of upstream kinases such as DNA-dependent protein (DNA-PKcs), Ataxia Telangiectasia Related protein (ATR) or Ataxia Telangiectasia
Mutated protein (ATM). The activity of these enzymes then leads the cell towards the first growth arrest stage.[20]
Figure 1.3 The role of telomere length in the development of cancer cells. Adapted from various references.[5, 19]
Telomeres shorten with every cell division.
Premalignant cells can bypass M1 checkpoint by transforming mutation, e.g. p53 and p16 inactivation.
M1 senescence M2 crisis
Self-renewal
Mean telomere length (kbp) 8
~5
~1 - 3
Cell doublings
0 ~30 - 70 ~70 - 90
Telomerase activation immortalises growth- deregulated cells.
Healthy cells
7 Premalignant cells, however, have acquired a number of oncogenic mutations that enable the cells to escape the M1 checkpoint, by deactivating tumor suppressor genes such as p53 and p16.[6, 29] The proliferation rate of premalignant cells also decreases as their telomeres shorten, leading eventually to mortality stage M2 (the second growth arrest or crisis stage). Entry of cells to this stage is followed by rampant genetic instability, merging of chromosome ends and extensive cell death.[5] As a consequence the majority of premalignant cells are restrained from proliferating further at this point, but on some occasions a cell can bypass this checkpoint and become effectively immortal. This generally involves the upregulation or reactivation of telomerase, or in much rarer cases, the alternative lengthening of telomeres (ALT) pathway which involves homologous DNA recombination mechanisms.[18, 25] Irrespective of the exact mechanism, most cells which escape the M2 crisis point have stable but short telomeres, as well as excessive telomerase activity. These features are now considered to be the major hallmarks of cancer.[5]