Crystal Nevins

 

Abstract

Telomeres are highly conserved, specialized nucleotide sequences located at the ends of chromosomes consisting of several hundred tandem six G rich nucleotide repeats in the 5’ to 3’ direction. Telomeres play several important roles in human cells including protection from degradation and homologous recombination. As cells duplicate, the telomere length shortens with each round or replication; this leaves chromosomes less protected over time. In humans, telomere shortening rates have been determined to be 20-60 bade pairs per year, depending on the tissue type. Telomeres have a dynamic structure and function, as it changes as cells age. There is a characteristic difference between telomeres found in human somatic cells and those found in cancer cells; there are many applications for cells with active telomerase in research and cancer therapies. This review outlines the structure and formation of telomeres and discusses their function as cells age. The role of telomeres in cancer cells will also be reviewed.

Telomeres are located at the ends of linear chromosomes functioning to protect their ends. Telomeres consist of several hundred tandem G rich nucleotide repeats in the 5’ to 3’ direction; in humans this sequence is AGGGTT. The length of telomeres is variable based on tissue type [1]. Telomeres can form a telomeric loop (T-loop) at the terminus of linear chromosomes providing more extensive protection [2].

In somatic cells, telomeres are shortened with each round of cell division. As the telomeres shorten, the ends of the chromosomes become more and more vulnerable [3]. Shortening of the telomeres, however, does not occur in most cancer cells. Cancer cells demonstrate telomerase activity throughout their lifespan allowing, the cells to become “immortal” [4]. While all cells contain telomerase, it is typically only active during development, more specifically during the G1 phase [5].  Telomerase will only remain active in cells that also express active human telomerase reverse transcriptase (hTRT) [6]. Better understanding the role of hTRT in cancer cells can lead to more advanced cancer treatments; hTRT and other associated proteins can be targeted for drug therapies specifically as it is not active in other somatic cells [7].

Telomeres

Telomere Function and Structure The main known function of telomeres is to protect linear chromosomes from degradation at their terminus. T loop structures form at chromosome ends by looping a 3’ overhang left on the end of the chromosome back and allowing it to become imbedded in the double stranded portion of the chromosome. This process is mediated by the telomeric repeat-binding proteins, TRF1 and TRF2 [8]. TRF1 and TRF2 are included in a complex called the shelterin complex. It consists of TRF1, TRF2, TIN2, Rap1, TPP1 and Pot1 and it functions to protect the ends of chromosomes by forming and maintaining telomeres [9].

TRF2 acts to sequester the single stranded end of telomeres and embed it within a double stranded section of the telomere. This results in a triple stranded structure at the point of insertion which forms a small displacement loop (D loop) [10]. Data shows that TRF2 is localized to the D loop junction and TRF1 is found at the double stranded portion of the telomere. While TRF2 has been classified as a double stranded telomere binding protein, experimental data shows that overexpression of a dominant negative TRF2 cell line has an effect on the singe stranded portion of the telomere. It also led to the activation of the p53 apoptosis pathway [8].

During DNA replication in somatic cells, polymerase is unable to replicate the 3’ end of the lagging strand of DNA (Figure 1. a-c). As a result, with each round of replication your chromosomes are less and less protected [3]. Associations have been made between telomere length and the age of chromosomes [11]. With each round of replication, between 20 and 60 base pairs in the telomere are lost; this number is dependent on tissue type [11].

Further functions include the prevention of homologous recombination at the ends of linear chromosomes [3] and the prevention of the activation of premature apoptosis [8]. Work done by Griffith, et al. show that depletion of TRF2 leads to, among other things, activation of a double strand break checkpoint. This checkpoint signals the p53 pathway which often leads to apoptosis. All of this together suggests the necessity of telomeres for maintaining the integrity of chromosomes [8].

T-Loops T-loops were discovered in the Griffith and de Lange laboratories in 1999 changing the way that telomere structure was previously understood. It was previously believed that chromosomes terminated in a 3’ overhang with a GT rich repeat. Klobutcher et al. provided the first indication that there is conservation among various species at chromosome ends; they recognized that there is varying length between species but that all contain the same 3’ overhang on the G rich strand [12]. The current understanding of telomere function was discovered in the Griffith lab in 1999. Using electron microscopy, the end structure of chromosomes was visualized as a loop.  Griffith et al. describe a three stranded displacement loop (D-loop) which folds over allowing the single stranded overhang to tuck into the double stranded structure. This process is accomplished with sequestering proteins, TRF1 and TRF2 [8].

TRF1 and TRF2 sequester the single stranded 3’ end of the telomere and loops it back to form a T-loop. While TRF1 is acting to align double stranded sequences of the T loop, TRF2 closes the loop by inserting the single strand into a section of the double stranded section of the telomere and forming a D loop structure [8].

Telomerase

The Discovery of Telomerase Telomerase was first discovered by Carol Greider and Elizabeth Blackburn in 1985. Greider and Blackburn first described telomerase as telomere terminal transferase and were later able to characterize it as an RNA ribonucleoprotein [10] [13].  The discovery of telomerase opened the field for new research; not long after the initial discovery in Tetrahymena thermophile, Jack Szostak discovered telomerase activity in human HeLa cells. HeLa cells were isolated from the ovarian cancer patient, Henrietta Lacks, several years prior and coined the immortal cells. Until this time, it was unknown what made these cells immortal. This era was the beginning of a new understanding of cell aging [4].

In 2009, the Nobel Prize in Physiology or Medicine went to Elizabeth Blackburn, Carol Greider and Jack Szostak for their work on telomere function and the mechanism of telomerase [14].

Regulation of telomerase Telomerase is a ribonucleoprotein complex containing a telomeric RNA subunit (TR), a catalytic core, and telomerase reverse transcriptase (hTRT). It is also surrounded by several accessory proteins [14].The TR component of telomerase is an RNA sequence complimentary to the repeated sequences found in the telomere; in humans this sequence is 3′-CAAUCCCAAUC-5′ [15]. Interestingly, only 6 nucleotides in this sequence are repeated in telomeres, the others act to orient the telomerase on the DNA. The RNA template sequence functions as a primer on the 3’ overhang of the lagging strand during DNA replication (Figure 1. d). The telomerase moves step wise in the 5’-3’ direction, continually adding repeats of 6 nucleotides long (Figure 1. e). Once this step is complete, DNA polymerase α is able to complete lagging strand elongation (Figure 1. F) [1].

The catalytic subunit of telomerase in Homo sapiens is Human telomerase reverse transcriptase (hTRT) [6]. Nakayama et al. characterized hTRT by measuring telomerase activity in human fibroblast cells overexpressing hTRT. They were able to see that when hTRT is overexpressed in telomerase negative cells, telomerase activity is induced. In cells containing  hTRT mutations, telomerase activity iss not induced thus characterizing the relations ship between hTRT and telomerase activity. This led them to measure activity in cancerous liver cells and non-cancerous liver cells where they saw consistent results. They came to the conclusion that hTRT is the catalytic protein responsible for activating telomerase and that it plays an important role in the immortality of cancer cells [6].

Telomeres and aging

The p53 tumor suppressor protein is responsible for imposing apoptosis and cell cycle arrest. As telomeres shorten critically to unsafe lengths, p53 is activated and senescence is promoted. Senescence is when cells no longer replicate, eventually leading to apoptosis [16]. This mechanism allows cells the ability to maintain the integrity of their chromosomes. Those with hyper shortened telomeres lose their protection and are subject to translocations, deletions, homologous recombination, and other damage [7]. This kind of damage can lead to tissue aging and degenerative disorders [16].

Some aging phenotypes in humans can be contributed to shortened telomere lengths, as shown in telomerase knockout mice. Telomerase knockout mice in late generations show signs of aging including graying fur and the inability to respond to physical stressors such as wound repair. Many of the phenotypes seen correlated with skin; internal organs including kidneys, brain, and cardiovascular system do not show advanced aging with shortened telomeres [18]. While this information shows promise in the field of telomeres and aging, it cannot be directly applied to humans. Mouse telomeres range from 50-150 kbp while in humans, they are about 15 kbp in length. This, combined with the short lifespan of mice in comparison to humans [17], suggests that the instability of chromosomes with shortened telomeres is likely more dependent on the vulnerability than their actual shortened length. Less telomeric protection at exposed chromosome ends can lead to recombination and cancer.

Aged chromosomes with shortened telomeres have a correlation with abnormalities such as Robertsonian fusions (end to end fusions).  They are also correlated with spontaneous tumor generation. Cancer generation was observed in telomerase knockout mice; there was a significant correlation in tumor generation in tissues that have been proven telomerase dependent such as the skin and testes [18].

Telomeres and Cancer

Telomerase activity in cancer cells The majority of cancer cell lines have activated telomerase activity giving them an extended, and sometimes immortal, life [4]. An estimated 80% of tumors contain active telomerase throughout their lifespan. It was recently shown that these telomerase positive tumors also express contain long interspersed nuclear elements-1 (LINE-1) which are responsible for telomere maintenance in pathological cells [19]. cMyc and Krüppel-like factor-4 (KLF-4) are potential oncogenes; cMyc codes for a transcription factor while KLF-4 is a transcription factor itself. When Aschacher et al performed LINE-1 knockdown, they observe increased telomere dysfunction and decreased levels of cMyc and KLF-4 were also knocked down. This suggests that LINE-1 acts as a regulatory element by means of cMyc and KLF-4 [19].

Cancer cells as research tools The research done by Aschacher et al. proposes LINE-1 as a rational target for inhibiting telomerase activity in cancer cells. Their experimentation shows that G2 cell cycle arrest is observed in LINE-1 depleted cell lines. This suggests that inhibition of the effects of LINE-1, either by directly targeting it or through cMya and KLF-4 , could be an effective way of repression tumor growth as LINE-1 is only found in active tumor cells [19].

Another proposed mark for cancer drugs is TRF2. TRF2 has shown to be essential in the formation of telomeres [8]. TRF2 disruption can impair telomere maintenance and elicit a DNA damage response. A chemical inhibitor is being studied to bind to the TRF2 domain to switch off its signaling [20].

Conclusion

The future of research regarding telomeres and cancer research lies in using associated proteins as drug targets. As telomerase activity is noted in 80% of tumor cells, telomerase and its associated proteins are a good place to start. It is a specific target as most adult somatic cells do not have active telomerase [21].  The downfall of this method is that existing cancer cells with elongated telomeres will have to go through significant numbers of cell division until senescence is reached [22].

References

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