Telomere Biology

Telomere Bio & Cancer PaperTelomere Biology and CancerIntroductionTelomere biology is an area of heavy research in hopes of finding a cure for cancer. Cancer is known to bypass regulatory systems during cell proliferation which allow the cells to divide uncontrollably. According to the review The Role of Telomere Biology in Cancer, telomeres and the enzyme telomerase play a significant role in the development of cancer. In this review, a variety of ways in which telomere biology can be used to initiate and possibly develop treatments for cancer are presented and evaluated. The survival of a species necessitates genomic stability: it ensures that all required information is passed on from one generation to the next, and so on. Eukaryotes require a complex and efficient DNA repair mechanism, which is time-sensitive in its onset and duration. Eukaryotic genome is organized in a linear fashion a configuration that lends itself to the problems of tail-end maintenance, and the need to differentiate these tail-ends, called telomeres (literally: the ending part), from DNA double-strand breaks (DSBs). DSBs are routinely employed in initiating cell death in cancer treatment. Telomeres are found at ends of linear chromosomes and are an extension of the parent DNA strand which allows for DNA polymerase to complete the lagging strand during DNA replication. DNA polymerase has a DNA primase subunit that enables it to supply its own primer. After applying the primer to the telomere, the polymerase can then finish the sequence. Preserving DNA is important since the loss of genetic information can lead to apoptosis of the cell or disease, such as cancer. There are a handful of mechanisms that support the integrity of telomeres. The enzyme telomerase is responsible for retaining the length of telomeres which are shortened after each round of cell division. If telomeres are too short or too long, this may lead to senescence, apoptosis or cancer. The latter has become evident in fast growing cells which trend to have high levels of telomerase expression (2). DNA-damage response proteins as well as other protein complexes aid in protecting telomeres. For instance, DNA-repair proteins along with the shelterin complex the formation of t-loops of telomere ends during G2 phase of cell cycle (3). These t-loops are believed to further protect the telomeres from damage by concealing the end of the DNA. However, if any of these proteins were to falter; this could lead to a loss of telomere signaling, an increase in telomere-free chromosomes or the proteins themselves may inhibit telomerase (3).Telomeres are a very elaborate arrangement: they are nucleoprotein complexes that physically cap the chromosomes ends and vary in length between 5 to 15kb in humans. It has a DNA repeat sequence of 5 TTAGGG 3 hexamer (referred to as the G-tail; outwards from the centromere) that overshoots its complimentary AATCCC sequence. This overhang bends on itself to form a lariat-like DNA loop (t-loop), while the 3 G-end forms a d-loop by extending into the t-loop. Thus, a secondary DNA structure is constructed, which inherently prevents the G-tail from being recognized as a DNA DSB and prevents access to DNA damage response (DDR) enzymes and nucleases. An almost cardinal rule of DNA replication is incomplete replication of the lagging strand, followed by post-replicative degradation of the 5 strand. This causes telomeres to be shortened after every cell cycle. Successive cell replications (aging) result in the inability of telomeres to form a t-loop, triggering chromosome fusion, exonuclease degradation, and/or DDR.There are a variety of DNA lesions and cells initiate a DDR via a signal transduction mechanism. DDR regulates transcription and gathers and deploys respective DNA repair apparatus to these lesions in response to metabolic conditions, and manages cell fate decisions by introducing cell cycle checkpoints. The eventual goal for DDR is to maintain genomic integrity by preventing replication of cells with damaged DNA. Deficiencies in DDR increase the predisposition of cancer and neurodegeneration.Of these DNA lesions, the DSBs have particular significance while they are readily caused by ionizing radiation, they have many endogenous sources: exposure to free radicals during DNA replication, errors made by the enzymes involved, and limited supply of certain nucleotides, enzymes, cofactors, and other proteins. Different types of chemical moieties are associated with DSBs that differ from the regular 5 phosphate and 3 hydroxyl ends allowing a wide variety of enzymatic activity to be available to prepare DSBs for repair; however, they may not be able to perform their requisite repair tasks as the complexity of the DNA lesions increase. In these events, nucleases are employed to cleave the damaged DNA sequences. Cells have developed five pathways for DNA repair. DSBs, which arise from single-stranded DNA (ssDNA) replication and free radicals, are repaired through homologous recombination (HR) and non-homologous end joining (NHEJ). Single-stranded breaks (SSBs) are repaired via three excision repair pathways: mismatch repair (MMR), nucleotide excision repair (NER), and base excision repair (BER). When a DSB is detected, the cascade of events is triggered by ATM (ataxia-Telangiectasia-Mutated), ATR (ATM and Rad3), PIKK (phosphatidylinositol-3-OH-kinase-like kinases), and DNA-PK (DNA-dependent protein kinase). During late G2 and S phases, availability of a second DNA copy triggers HR, and while the repair is error-free, it requires the action of additional proteins Replication Protein A (RPA), DNA dependent ATPase RAD 54, strand exchange factor RAD51, and BRCA1-BRCA2 proteins. NHEJ is triggered when a sister chromatid is not available as a template (during G1 and early S-phase). This entails direct end-ligation and results in either gain or loss of a few nucleotides. The Key Players and Factors in the Telomere Story (vis--vis Cancer)In mammals, there are six dedicated telomere-specific proteins: Telomeric Repeat Binding Factor 1 (TRF1), Telomeric Repeat Binding Factor 2 (TRF2), TRF2 Interacting Protein (RAP1), Adrenocortal Dysplasia Protein Homolog (TPP1), TRF1 Interacting Nuclear Factor 2 (TIN2), and Protection of Telomeres 1 (POT1). Collectively, these are the shelterin complex. TRF1 and TRF2 bind directly to the telomere double strand, while POT1 binds to the single stranded telomeric overhang and protects the 3 G-tail. TPP1 forms a complex with POT1 while binding with ssDNA, and may also be involved in recruiting telomerase. TIN2 links ssDNA and dsDNA binding complexes, most probably at the d-loop formation. RAP1 is the most conserved subunit of the shelterin complex, which binds to TRF2 and also acts as a transcription regulator. TRF1 and TRF2 bear similar domains recognizing TTAGGG domain on dsDNA of the telomere and has a docking motif TRFH (TRF homology) that dimerizes either TRF1 or TRF2. This shelterin complex is located at the end of every telomere and helps stabilize the isolated secondary structure of the DNA at the G-tail, while preventing recognition by DDR. As such, the shelterin complex envelops chromosome ends from DNA damage-sensing and repair pathways. It also regulates post-replication restoration of telomeric DNA and acts as a platform for DNA replication, recombination, and remodeling of the chromatin. Another protein complex called CST contains 4 subunits: DNA polymerase , two primase accessory factors CTC1 (CST telomere maintenance complex component 1) and STN1 (suppressor of cdc thirteen 1), and TEN1 (telomeric pathways with STN1). The CST complex has been found to associate with only a subset of the telomeres. While research is still being conducted, the CST complex is probably involved in limiting overhang length and in the synthesis of the telomeres C-strand (complimentary to the G-tail), and in cell replication by releasing stalled replication forks. The telomere nucleoprotein complex has a third non-coding RNA component called TERRA (telomeric-repeat-containing RNA) and is thought to act as a direct telomerase inhibitor and important for telomere maintenance and function. TRF2, via its G-quadruplex structure (G4), binds TERRA, believed to introduce another level of regulation. TERRA forms an integral part of telomere heterochromatin and is involved in the state of telomeric chromatin as the cell develops and differentiates. Transcription of TERRA occurs at almost all chromosome ends and occurs in response to telomere length. TERRA expression varies during the cell cycle, with a low level in the late S phase and a high level in early to mid G1 phase. Some studies have found interference with telomere replication as TERRA accumulates. Telomerase is a specialized multisubunit ribonucleoprotein (RNP) that elongates telomere sequences onto chromosome ends. It has two conserved subunits: TERT (telomerase reverse transcriptase) containing the catalytic subunit, and TERC (telomerase RNA component), which together form a complex that recognizes the 3OH at end of the G-tail overhang and, using TERC as a template, elongates the telomere. It also contains protein dyskerin (DKC1), which in-turn forms a core complex with three smaller RNPs NH2, NOP10, and GAR1 (Gly-Arg Rich domain). Together with NOLA1 and NOLA 3 proteins (not identified in the figure), these catalyze pseudouridylation (isomerization of uridine where ribose is attached to C5, not N1) of rRNA, believed to be required for correct processing or trafficking of TERT. For the telomerase (holoenzyme in humans) assembly, well-conserved ATPases pontin and rentin are also required. These proteins also play an important role in chromatin remodeling, SUMO modification, and transcription regulation. When the pontin/reptin association with TERT complex was treated with DNase1, ethidium bromide, and RNAase A in HeLa cells, they were found to be highly resistant, indicating copurifying nucleic acids dont play a significant role. Holoenzyme telomerase is expressed in the early stages of embryogenesis which is then repressed in almost all somatic cells, except male sperm cells, lymphocytes, and any stem cells. The TERT-TERC-DKC1 (mature telomerase) complex has high enzymatic activity as compared to the TERT-pontin-reptin (pretelomerase) complex that has a much lower catalytic activity. Formation of the pretelomerase complex is dependent on cell cycle, present in the S phase and degrades in the G2, M, and G1 phases. Evidence also suggests pontin-reptin can chaperone TERT into different complexes that allows it to activate dormant epidermal cells. Cancer cells end up reactivating telomerase. Regulation of the telomerase is conducted at TERT transcription, which in turn is regulated by oncogenes and tumor suppressor genes. Telomerases are transported through Cajal bodies, which are involved in RNP complex biosynthesis. This transport is regulated by telomerase Cajal body protein 1 (TCAB1). Telomeric DNA in mammals is compiled into equally spaced nucleosomes that carry epigenetic marks reflective of their constitutive heterochromatin. The factors that affect their heterochromaticity methylation of histones, acetylation change the conformation of these chromatins resulting in increased telomere length. This becomes increasingly important in the incidence of cancer in humans where there is a significant dearth of telomerase.When Telomeres and Telomerases go AwryIn the absence of a telomeres protective mechanism, through disruption of the shelterin components or normal attrition due to replication, causes the ends of chromosomes to masquerade as a DSR, triggering the cells DDR. It has been found that ATM and/or ATR help coordinate the action of a DDR on a dysfunctional telomere. As TRF2 depletes, it triggers ATM. ATR is triggered either by POT1 degradation (3 telomeric overhang is deprotected) or TRF1 depletion. When upstream DDR is dysfunctional, tumor-prone phenotypes may emerge. Loss of ATM function causes Ataxia-Telangiectasia (A-T), a neurodegenerative disease that affects the nervous system, immune system and some organs.Dysfunctional telomeres are highly interesting, as they can trigger either of opposing pathways in promoting genome stability or compromising it. Replicative senescence a basic feature of somatic cells whereby cells alter the expression of a few growth-regulatory genes leading to cellular arrest, limiting the number of divisions they can experience. This is an important mechanism which cells adopt to prevent proliferation of potential cancer cells. Senescence inducing stimulus stems from the loss of telomeres which triggers a persistent DDR that maintains growth arrest. Other senescence stimuli include HDAC (histone deactylase) inhibitors, which help open up the chromatin, activate ATM and the tumor suppressor p53. Oncogenes deliver a strong mitogenic signal causing errors in DNAs replication of origins and replication forks, whereby cells experience senescence, and triggers a persistent DDR. However, cells can also senesce without a DDR being triggered (mechanism not clearly known). The propensity of malignant cancers to occur and proliferate increases with age. The incidence of senescent cells increases with age in a variety of tissues, and increasingly so in mitotically-able tissues. These very tissues, in-turn, help trigger cancer cells. Senescent cells also develop a secretory phenotype (SASP) and much of what they secrete (e.g. growth-related oncogene, cytokines interleukin-6) is associated with aggressive cancer cells. In TERT-null mice, there was cytogenetic evidence of telomere dysfunction, which caused these mice to have a shortened life-span and multi-organ degeneration. In a yet different study, p53-null mice were found to develop cancer as telomere dysfunction progressed, which was inhibited by DDR. TERT expression caused the DDR to be inhibited and allowed the progression of tumors. Introduction of TERT into mature human fibroblasts allowed them, it was found, to bypass senescence and become immortalized.The ability for the telomeres to protect the DNA greatly depends on the proper function and binding of each component of the shelterin complex. A malfunction of the protein can lead to end-to-end telomere fusion or recombination between sister telomeres. The incidence of tumorous growth, when evaluating colon and breast tissue, has demonstrated an increase in fusion and recombination. In breast cancer, while analyzing both tissue samples and cell cultures, there is support for telomere degradation leading to an outgrowth of cancerous cells where telomerase is present. There have been conflicting conclusions from various studies evaluating telomere attrition some determining that decreased telomere length is associated with higher cancer risk, a longer length is associated with increased cancer risk, and some no association between length and cancer risk. While these studies do employ different methodologies, it is clear that the telomere role in cancer propagation or prevention is context dependent.In human cancer, telomere dysfunction is involved in early chromosome instability, long-term cellular proliferation, and possibly other processes related to cell survival and microenvironment. Telomeres constitute an attractive target for the development of anti-cancer drugs. In particular, individual protein components of the shelterin complex are promising candidate targets for cancer therapy.Other Mechanisms by which Telomerase impacts CancerIt has been established that an increased presence of telomerase increases the incidence of tumors. This is accomplished by preventing telomere attrition, thereby promoting cell division. In experimentation, activation or over-expression of telomerase is achieved by an over- or under-expression of TERT. Cell changes that are rapidly manifested are evaluated those that occur prior to any perceived impact on telomere length or function. Some strategies involve the introduction of TERT mutants, some that lack catalytic activity (D868A) or that are unable to maintain telomere length. Catalytic ability was found to encourage cell proliferation and limiting apoptosis in mitogen-deficient medium, thus enabling cell growth. TERT was found to be present in significant quantities in the mitochondria and impacts mitochondrial function in yet unknown mechanisms. When exploring catalytic ability, it was discovered that TERT proteins form complexes with RNA components, instead of TER, creating double-stranded RNA that create siRNA which essentially interfere with replication. There are multiple challenges with evaluating the catalytic component completely (variety between mammals, speed of catalytic activity, etc.) complicating the ability to understand physiological impact. TERT mutated for lack of catalytic activity has shown the induction of hair follicle stem cells in mice, in the absence of TER. A recent area of focus, these TERT D868A mutants have yet to arrive at meaningful inferences. Alternative Lengthening of Telomeres It has been observed in HeLa cells that even in the absence of telomerase, there is an appreciable level of telomere length maintenance being exercised. Telomerase-negative cancers adopt a variety of different mechanisms to maintain telomere length, referred to collectively as ALT. These are usually found in pancreatic tumors, sarcomas, and brain tumors. ALT cells contain a wide range of telomere lengths from the extremely long to the extremely short. The incidence of recombination at telomeres, as well as interchromosomal telomere recombination has been found to be very high. T-SCE levels are also elevated. There is a big overlap between the proteins required for recombination and for telomere maintenance in these cells. ATRX, a chromatin remodeler, mutations in which lead to mental retardation and facial and genital abnormalities, and DAXX (death-domain associated protein) a promoter of apoptosis, associate with each other to deposit a histone variant at specific chromatin regions. Somatic mutations in this complex have been identified in pancreatic and brain tumor cells. These mutations can be either frameshift or missense, and have been found to be present in most tumors. This is still an active area of research, but current studies reveal a causation with repressed telomeric heterochromatin state which activates recombination of telomeres and triggers ALT.Telomere-based Therapeutic Strategies

Our evaluation of papers resultsAfter reading this article, we had a much better understanding of why a cure for cancer is so difficult to discover.

The telomere biology of mice is different then that of human telomere biology, how can we conclude that the results from the experiments with mice are relevant to human biology??

ConclusionThe study of telomere biology has enhanced our knowledge of the origin of cancer and has given us possible avenues to explore for inhibiting its progression. However, these studies are far from complete! Every possible tactic has its pluses, minus and unknown side reactions. Studies have shown that the enzyme telomerase is a key player in cell proliferation and that its activity is increased by 85% in human cancers (3). Therefore, the inhibition of telomerase activity seems to be a likely option for a therapeutic strategy; however, four potential problems arise. First, in the absence of telomerase, the telomeres become too short for DNA polymerase to duplicate the ends of the chromosomes. The telomere depleted chromosomes then trigger a DNA-damage response which usually results in senescence. Even though the cells may not be dividing any longer, they may secret a toxin that causes inflammation which later may result in cancer (3). Secondly, the cancer cells could bypass regulatory mechanisms via ALT pathways and facilitate the growth of cancer! Thirdly, there is the uncertainty that all cancers will react the same way to the telomerase inhibitor, plus, there could be unforeseen side affects. And lastly, there is the issue of how to administer the inhibitor to just the targeted cells. If the cancer formed due to a depletion of the telomere, then, the healthy cells with telomerase activity may survive. Conversely, what if the cancer was caused by the telomeres being too long, then this approach would be irrelevant. Another possible therapeutic strategy would be to disrupt the function of the telomere itself. Even though this approach may be quicker then waiting for the telomere to shorten, this method faces similar dilemmas as with using a telomerase inhibitor; such as, toxicity, the possible use of ALT pathways and targeting administration. The telomere-disrupting agents would not be able to distinguish between healthy and cancerous cells; therefore, the survival of normal cells would be affected as well. A third option discussed was the hopeful strategy with the use of telomerase-targeted immunotherapy. Cytotoxic T lymphocytes (CTL) can recognize antigens and TERT determinants on the surface of cancer cells and destroy them; however, this method cant be used to target all types of cancer. The tumor must express a tumor antigen or telomerase in order for CTL to recognize it, plus, a wide range of antigens would be needed to target different cancers.The last therapeutic option mentioned was the use of oncolytic viruses which prohibits the replication of TERT-positive cells. Not only does this method have concerns with the survival of healthy cells, but a better understanding of whether or not TERT has other cellular functions besides telomere lengthening is required. Unknown adverse effects may be associated with the disruption of TERT.Telomere biology is important to ensure genome stability; however, encouraging telomere dysfunction may inhibit the formation of cancer. Before a cure for cancer can be discovered, continued research is most definitely required for a more comprehensive understanding of telomere biology.

Bibliography1. "Ataxia-telangiectasia." Genetics Home Reference. 2 Mar 2015. U.S. National Library of Medicine. 3 Mar. 2015. .2. Berg, Jeremy M., John L. Tymoczko, and Lubert Stryer. Biochemistry, Seventh Edition. New York: W.H. Freeman and Company, 2012. pg. 819-846.3. Xu, Lifeng, Shang, Li, and Bradley A. Stohr. "The Role of Telomere Biology in Cancer." Annual Review of Pathology: Mechanisms of Disease. Annual Reviews, 28 Aug. 2012. Web. 14 Feb. 2015. .

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