1.4 Telomerase

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Telomerase Biochemistry

 

Immortalization is one of the defining features of cancerous cells. Telomerase activation is the most common method of immortalization, allowing tumors to bypass the intrinsic shortening of telomeres that occurs during the normal process of DNA replication (1). In normal cells, successive rounds of cell division shorten the telomeres to the point where genomic instability and activation of DNA damage machinery, like the tumor suppressor p53, trigger apoptosis or replicative senescence (1). In this section, we will explain the major components and functions of telomerase, and how it is activated in cancerous cells.

 

 

Telomerase Structure and Activity

 

The telomerase holoenzyme is comprised of several major subunits: RNA-dependent DNA polymerase (TERT), telomerase RNA (TERC), and TERC-binding proteins, which stabilize TERC and promote holoenzyme assembly (2). TERT is the only highly conserved component of the complex (2). Human telomerase is organized as a dimeric complex, with both TERT subunits working together to yield catalytic activity (3). If one of the two TERT subunits in a dimer is nonfunctional, overall catalytic activity of the holoenzyme is greatly reduced (3). The catalytic TERT subunit is a reverse transcriptase responsible for synthesizing telomeric DNA through the addition of nucleotides to the DNA strand that is exhibiting the 5’ to 3’ orientation, elongating the 3’ overhang. When active, TERT binds TERC and uses the RNA sequence intrinsic to TERC (TTAGGG) as a template for DNA synthesis (2). The RNA component of TERC is highly complex, containing a template region, along with a number of other domains required for in vivo stability and activity (2). These domains include: a telomerase essential N-terminal domain, a TERT RNA-binding domain, a reverse transcriptase domain, and a TERT C-terminal extension domain (4). The template region of TERC binds to the 3’ G-strand overhang of telomeres and is responsible for guiding DNA synthesis (2). The secondary structure of the other regions is important in maintaining enzyme stability and function, reducing telomerase activity and processivity when mutated (2).

When activated, telomerase lengthens telomeres. This cycle has two important steps involving telomerase. During the first stage of telomere synthesis, the TERC template binds to the G-strand overhang and initiates synthesis of a new telomeric repeat (2).  This is called elongation. The speed of nucleotide addition is known as type I processivity. After synthesizing a telomeric repeat, the enzyme either terminates the reaction or shifts down the DNA strand to elongate the telomere through tandem repeats (2). The exact process of translocation is not well understood. The rate of template translocation and annealing is known as type II processivity, and is the rate-limiting step in telomere synthesis (2). Evidence now suggests that presence of the POT1 and TPP1 proteins increase type II processivity, perhaps regulating the process of translocation (2). Once a sufficiently sized 3’ overhang is constructed, the complementary strand is synthesized by DNA polymerase from the cell’s own replication machinery to complete the double stranded telomere.

Here is a very informative video which presents both the end replication problem and the action of telomerase in a clear way.

 
 

Telomerase Activation in Cancer

 

Typically, telomerase is active only during the S-phase of the cell cycle, allowing a small number of normal cells to maintain telomere length (5). Its expression has only been shown to occur in stem cells, leukocytes, skin cells, cells of the intestinal epithelium, and reproductive tissues. Nevertheless, 80-95% of tumors maintain high levels of telomerase activity in nearly every cancer of the body (5). Mutations in the genes encoding TERC and TERT are frequently responsible for the altered telomerase levels in human cancers. The copy numbers of these genes are frequently amplified in cancers, resulting in an increase in active enzyme (5). Changes in histone modification patterns, such as methylation/acetylation, also appear to play an important role and can dramatically alter TERT expression (5). Many transcription factors and signaling pathways can activate TERT gene expression, and several of these have been shown to also activate the TERC gene (5). The TERT and TERC genes can be activated in a multitude of different ways, which may explain telomerase's involvement in the majority of human cancers.

 

Subcellular localization might also play an important role in telomerase regulation and activation in transformed cells (6).  GFP-tagged hTERT proteins displays a nucleolus-to-nucleoplasm relocalization that coincides with the expected timing of telomere replication in wild-type cells.  This compares to transformed cells where normal regulation is disrupted and telomerase is compartmentalized to the nucleoplasm throughout the cell cycle (6).  This suggests that telomerase may be regulated in normal cells through its subcellular localization, and confining it to the cytoplasm will limit its exposure to DNA, therefore limiting its enzymatic activity.

 

Detection of Telomerase Activity

Measurement of telomerase activity can serve as a proliferation marker, especially in a majority of tumours that have increased telomerase activity. The ability to measure telomerase activity is also important in the search for telomerase inhibitors for potential anti-cancer drugs (11).  It has also been suggested in the literature that telomerase activity detection assays can be divided into two groups; one groups being based on detection of telomerase products and one based the amplification of DNA yields due to telomerase activity(11).

 

Fast and efficient detection of telomerase activity is most commonly done by TRAP (telomeric repeat amplification protocol) assays. These assays are PCR-based and allow for more uniform extraction of telomerase from small samples. Generally, the assay is done in a single tube and the first step is telomerase extension of the oligonucleotide trimer (TS), which is a substrate for the telomerase in the tissue sample. The second step is PCR amplification of the product using a forward oligonucleotide primer TS paired the reverse primer (CX) (10). These primers consist of non-telomeric sequence as well as the telomeric hexanucleotide sequence.  In the presence of deoxyribonucleotide triphosphates (dNTP’s) and telomerase in the cell lysate, the primers will extend the telomeres with the aid of Taq polymerase (12). Modifications in labeling of the PCR product for subsequent detection include the use of fluorescent, radio active or affine labels (11). The PCR product is then analyzed by gel electrophoresis. Analysis of the DNA molecules reveals differeing sizes that correspond to hexanucleotide increments. Included in these assays should be a negative control that determines if there are any heat resistant DNA polymerases present in addition to the telomerases. To accomplish this a portion of cell lysate is heat-treated to inactivate any of the telomerase activity(12). Variations and modifications of this assay are also used, such as the transcription amplification assay, scintillation proximity assay, hybridization protection assay, and the magnetic bead-based extraction assay(11). 

 

Telomerase Targeting in Anti-Cancer Therapeutics

 

Telomerase is over-expressed in up to 95% of human cancers and has a low expression level in normal non-transformed cells; therefore it is a promising target for anti-cancer therapeutics (7). Current telomerase-targeting therapies target telomerase activity by either interfering with the activity or binding of its catalytic subunit hTERT or telomerase's RNA template hTER (8). Inhibiting telomerase acitivy results in the resumption of telomere shortening during cell division, and restores the ability of the cells to undergo crisis and apoptosis triggered by a threshold telomere length (8). Tumour recurrence, a significant delay between treatment and results, and the development of resistance to telomerase inhibitors after telomerase shortening is common with anti-telomersae therapies (8). However, anti-sense oligonucleotides inhibitors that target either hTERT or hTER have shown promise as telomerase-targeted therapy as they do not lead to multi-drug resistance (8). Also, as telomerase is found in a large range of human tumours and immuno-tolerance against it is weak and incomplete, it is considered a universal tumour antigen and its peptides are capable of inducing a strong T-lymphocyte response in vivo (8). Treatments that stimulate hTERT-specific cytotoxic T cell lysis and promote a strong anti-telomerase immune response have the potential to reduce malignant tumour burden (8). Currently, vaccines are being developed that may promote immune recognition of hTERT and enhance its binding to MHC molecules (8).

 

Inhibition of hTERT as a monotherapy has shown little efficacy in clinical trials (9). An increase in gene activity of the oncogenic promoter COX2 was shown to occur when hTERT is knocked down in cancer cells. However, targeting both hTERT and COX2 has been shown to significantly reduce tumour growth in vitro and in mice (9). Combining anti-telomerase therapies with other oncogene-targeted therapies or standard chemotherapies is suggested to maximize treatment efficacy and improve prognosis (8).

 

 

References

  1. Mocellin, S., Pooley, K. A, & Nitti, D. (2013). Telomerase and the search for the end of cancer. Trends in molecular medicine, 19(2), 125–33. doi:10.1016/j.molmed.2012.11.006
  2. Rubtsova, M. P., Vasilkova, D. P., Malyavko, A. N., Naraikina, Y. V, Zvereva, M. I., & Dontsova, O. A. (2012). Telomere lengthening and other functions of telomerase. Acta naturae, 4(2), 44–61.
  3. Sauerwald, A. et al. (2013). Structure of active dimeric human telomerase. Nature Structural & Molecular Biology, 20(4): 454-460.
  4. Wu, X. et al. (2013). Feedback regulation of telomerase reverse transcriptase: new insight into the evolving field of telomerase in cancer. Cellular Signalling, 25: 2462-2468.
  5. Skvortzov, D. a, Rubzova, M. P., Zvereva, M. E., Kiselev, F. L., & Donzova, O. a. (2009). The regulation of telomerase in oncogenesis. Acta naturae, 1(1), 51–67. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3347505&tool=pmcentrez&rendertype=abstract
  6. Wong, J.M.Y et al. (2002). Subnuclear shuttling of human telomerase induced by transformation and DNA damage. Nat. Cell Biol, 4: 731-736
  7. Bisoffi, M., Heaphy, C.M., and Griffith, J.K. (2006). Telomeres: prognostic markers for solid tumours. Int. J Cancer, 119: 2255-60.
  8. Ruden, M., and Puri, N. (2013). Novel anticancer therapeutics targeting telomerase. Cancer Treatment Reviews, 39(5): 444-56.
  9. Liu, T. et al. (2013). Telomerase reverse transcriptase inhibition stimulates cycloocygenase 2 expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects.  British Journal of Cancer, 108: 2272-2280
  10.  Piatyszek, MA, Kim, NW, Weinrich, SL, Hiyama, K, Hiyama, E, Wright, WE, Shay, JW. 1995. Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods in Cell Science. 17:1-15.
  11. Skvortsov, D, Zvereva, M, Shpanchenko, O, Dontsova, O. 2011. Assays for detection of telomerase activity. Acta Naturae. 3:48.
  12. Weinburg, RA. 2007. The Biology of Cancer. Garland Science, Taylor & Francis Group, New York, NY.