Telomere Assignment

Telomeres Telomeres are tandem repeats ((TTAGGG/CCCTAA)n in vertebrates) that are found at the ends of linear eukaryotic chromosomes80. Their length varies between chromosomes and species, but can range from a few to tens of kilobase pairs. Telomeres have several roles: they prevent the ends of linear chromosomes from appearing as DNA breaks, they protect chromosome ends from degradation and fusion, they allow complete chromosome replication and they position the chromosomes within the nucleus.

Telomere structure Telomeres are, through necessity, dynamic structures. Although the ends of the chromosomes need to be camouflaged to avoid being seen as DNA breaks, DNA replication requires remodelling of this structure. The repetitive sequences of telomeres are bound by both duplex-DNA-binding proteins, which include TRF1 and TRF2 , and single-stranded DNA-binding proteins .

Telomeres end in a 3' single-strand overhang. The telomere end folds back on itself, forming a protective 'T-loop', to hide the vulnerable 3' overhang. This single-stranded DNA invades and hybridizes with a region of the doublestranded telomere repeat and the displaced DNA forms a small 'D-loop'. Telomere-binding proteins — for example, TRF2, which binds the T-loop juncture — are important in maintaining the stability of this structure (see upper figure).

Telomere shortening and the end-replication problem Telomeres shorten in part because of the end replication problem that is exhibited during DNA replication in eukaryotes only. Replication of the DNA lagging strand occurs by extension of primers in the 5' 3' direction. At the ends of the chromosomes, the terminal primer on the lagging strand (3' end) leaves a stretch of DNA that cannot be replicated. As a result of this, the telomeres shorten by 50–200 base pairs with each cell division. Telomerase is a telomere-specific ribonucleoprotein reverse transcriptase that adds single-stranded telomeric repeats to the chromosomal 3' ends, preventing continual telomere shortening. However, in vitro studies (von Zglinicki et al. 1995, 2000) have shown that telomeres are highly susceptible to oxidative stress. Telomere shortening due to free radicals explains the difference between the estimated loss per division because of the end-replication problem (ca. 20 bp) and actual

telomere shortening rates (50-100 bp), and has a greater absolute impact on telomere length than shortening caused by the end-replication problem

Telomerase structure Human telomerase consists of two molecules each of human telomerase reverse transcriptase (TERT), telomerase RNA (TR or TERC), and dyskerin (DKC1). The genes of telomerase subunits, which are TERT, TERC, DKC1, and TEP1 etc, are located on different chromosomes in the human genome. Human TERT gene (hTERT) is translated into a protein of 1132 amino acids. TERT proteins from many eukaryotes have been sequenced. TERT polypeptide folds with TERC, a noncoding RNA (451 nucleotides long in human). TERT has a 'mitten' structure that allows it to wrap around the chromosome to add single-stranded telomere repeats.

Telomerase function By using TERC, TERT can add a six-nucleotide repeating sequence, 5'-TTAGGG (in all vertebrates, the sequence differs in other organisms) to the 3' strand of chromosomes. These TTAGGG repeats (with their various protein binding partners) are called telomeres. The template region of TERC is 3'CAAUCCCAAUC-5'.[12] This way, telomerase can bind the first few nucleotides of the template to the last telomere sequence on the chromosome, add a new telomere repeat (5'-GGTTAG-3') sequence, let go, realign the new 3'-end of telomere to the template, and repeat the process. (For an explanation on why this elongation is necessary see Telomere shortening.)

ALT pathway of telomeres In most human somatic cells, telomerase activity is very low. This leads to gradual telomere shortening which, in turn, can trigger replicative senescence, a process where a cell with critically short telomeres permanently exits from the cycle of division. In contrast, the great majority of cancers are able to maintain their telomere lengths indefinitely. In most cases, this occurs because of an up-regulation of telomerase activity.

However, some cancers maintain their telomere lengths through a telomerase-independent process termed alternative lengthening of telomeres (ALT). The telomeres in ALT cells are highly heterogeneous, often extremely long and appear to be maintained through homologous recombination.

Telomeres , the Hayflick limit and Cell Immortality The Hayflick limit (or Hayflick Phenomenon) is the number of times a normal cell population will divide before it stops, presumably because the telomeres shorten to a critical length. The Hayflick limit was discovered by Leonard Hayflick in 1961, at the Wistar Institute, Philadelphia, when Hayflick demonstrated that a population of normal human fetal cells in a cell culture divide between 40 and 60 times. It then enters a senescence phase (refuting the contention by Alexis Carrel that normal cells are immortal). Each mitosis shortens the telomeres on the DNA of the cell. Telomere shortening in humans eventually makes cell division impossible, and it correlates with aging. This mechanism appears to prevent genomic instability and the development of cancer. In cultured cells, the loss of telomeric DNA depends on the number of cell divisions, and Allsopp et al. have noted that telomere length is a useful predictor of the residual proliferative capacity of cells . In this context, the question arises as to whether one could achieve unlimited replication capacity and immortality in somatic cells if telomere length could be maintained. Counter et al. transfected normal human embryonic kidney cells with Simian Virus 40 tumor antigen (SV 40 T), forming a tumor virus protein that extended the lifespan of cultured cells. The transfected cells divided and entered a point of crisis, in which most of the cells died; only some cells became immortal. During the period of cell division, the telomeres shortened continually and no telomerase activity could be detected. Those cells that survived the crisis point and became immortal had reactivated telomerase and stabilized their telomeres. This means that even somatic cells can gain the ability of endless replication if telomere length is maintained and (or) the enzyme telomerase is activated. Cells in culture are thought to stop dividing because of activation of an antiproliferative mechanism termed ―mortality stage 1‖ (M1). The stimulus for the induction of M1 may be DNA-damage signals from the altered expression of subtelomeric regulatory genes or from a critical shortened telomere. P53 and the retinoblastoma gene product pRb are involved in the execution of M1. One hypothesis for the induction of M1 postulates the following: (a) a single chromosome denuded of telomeric repeats produces a

DNA-damage signal, which (b) induces p53 and p21; (c) p21 inhibits the cyclin-dependent kinases, which then (d) are prevented from phosphorylating pRb; (e) the presence of unphosphorylated pRb coupled with other actions of p53 and p21 results in the M1 arrest (21). If these cell cycle regulators are mutated or blocked, the cells continue to divide and thus the telomeres continue to shorten. Cells divide until a second independent block in proliferation is reached, termed ―mortality stage 2‖ (M2). The M2 mechanism is probably induced when so few telomere repeats remain that the unprotected chromosomal ends block further proliferation. The M2 block might be overcome in some cells by reactivation of telomerase, the repair of chromosome ends, the stabilization of telomere length, and the generation of an immortal cell clone .

Telomeres , Stress and Aging

A major cause of aging is "oxidative stress." It is the damage to DNA, proteins and lipids (fatty substances) caused by oxidants, which are highly reactive substances containing oxygen. These oxidants are produced normally when we breathe, and also result from inflammation, infection and consumption of alcohol and cigarettes. In one study, scientists exposed worms to two substances that neutralize oxidants, and the worms' lifespan increased an average 44 percent. Another factor in aging is "glycation." It happens when glucose sugar from what we eat binds to some of our DNA, proteins and lipids, leaving them unable to do their jobs. The problem becomes worse as we get older, causing body tissues to malfunction, resulting in disease and death. This may explain why studies in various laboratory animals indicate that restricting calorie intake extends lifespan. It is possible oxidative stress, glycation, telomere shortening and chronological age - along with various genes - all work together to cause aging. Geneticist Richard Cawthon and colleagues at the University of Utah found shorter telomeres are associated with shorter lives. Among people older than 60, those with shorter telomeres were three times more likely to die from heart disease and eight times more likely to die from infectious disease.

It was also found that in patients with syndromes of accelerated aging [progeria (i.e., Hutchinson–Gilford syndrome) and Werner syndrome], the mean telomere lengths in cell cultures were considerably shorter than in normal individuals. These premature aging syndromes are characterized in progeria by growth retardation and accelerated degenerative changes of the cutaneous, musculoskeletal, and cardiovascular systems in young patients , and in Werner syndrome, for which recently the a candidate gene has been identified , by an early-onset and accelerated rate of development of major geriatric disorders such as atherosclerosis, diabetes mellitus, osteoporosis, and various neoplasms .

The new findings also suggest that chronic stress, and the perception of life stress, each had a significant impact on three biological factors -- the length of telomeres, the activity of telomerase, and levels of oxidative stress -- in immune system cells known as peripheral blood mononucleocytes, in healthy premenopausal women. At Harvard, they bred genetically manipulated mice that lacked an enzyme called telomerase that stops telomeres getting shorter. Without the enzyme, the mice aged prematurely and suffered ailments, including a poor sense of smell, smaller brain size, infertility and damaged intestines and spleens. But when DePinho gave the mice injections to reactivate the enzyme, it repaired the damaged tissues and reversed the signs of ageing .The key question is what might this mean for human therapies against age-related diseases? While there is some evidence that telomere erosion contributes to ageassociated human pathology, it is surely not the only, or even dominant, cause, as it appears to be in mice engineered to lack telomerase. Furthermore, there is the ever-present anxiety that telomerase reactivation is a hallmark of most human cancers."

Telomeres, telomerase, and cancer The cell cycle includes the orderly sequence of events that ensure the faithful duplication of all the cellular components in their correct sequence and the partitioning of these components into two daughter cells. Two classes of genes and their protein products are used to accomplish this process: genes whose products are obligatory for progress through the cell cycle phases, and genes whose proteins act as checkpoints for monitoring the efficacy and completion of these obligatory events and stopping the progression through the cell cycle if conditions are not satisfactory. The loss of cell cycle control generally leads to cell death but can also result in abnormal cells that continue to replicate and eventually form a tumor . The theory of carcinogenesis suggests that unlimited cell proliferation is required for development of malignant disease, and cancer cells must attain immortality for progression to malignant states. As shown above, shortening of telomeres may contribute to the control of the proliferative capacity of normal cells, and the enzyme telomerase may be essential for unlimited cell proliferation. The length of telomeres in cancer cells depends on a balance between the telomere shortening at each cell cycle and the telomere elongation resulting from telomerase activity. Tumors with shorter telomeres than in the original tissue have been detected in many cancer types . In neuroblastoma, endometrial cancer, breast cancer, leukemias, and lung cancer, a correlation between decreasing telomere lengths and an increasing severity of disease has been described. Short telomeres seem to be a primary cause for karyotype instability in malignant cells. According to the above-described theory of telomere dynamics during cell progression, tumor cells with

shortened telomeres can be considered to have undergone many cell divisions, with an accumulation of various genetic alterations. After a point of critical telomere shortening, telomerase might be reactivated to stabilize or elongate the telomeric DNA. Tumors with telomeres just as long as or even longer than in the original tissue seem to be rarer but have been described in some human malignant tissues, e.g., intracranial tumors, basal cell carcinomas of the skin, and renal cell carcinomas . There are two possible explanations for this phenomenon: Either an activated telomerase has elongated the once-shortened telomeres back to former length, or the tumor cells have not yet undergone enough cell divisions to induce significant shortening of telomeres. Telomerase is absent in most human somatic cells but, as Rhyu reports , was detected in 85% of 400 tumor tissue samples. Low amounts of telomerase activity in normal human tissues were found only in hematopoietic progenitor cells and activated T- and B-lymphocytes ; in germ cells, ovaries, and testes; and in physiologically regenerating epithelial cells . Results from examinations of normal tissues and benign cancers as well as malignant primary and metastatic tumors permit several conclusions. As in most normal tissues, telomerase activity is not expressed in somatic tissues adjacent to the tumor tissue. Accordingly, telomerase activity has proved to be a reliable marker for detecting tumor cells in resection margins. In benign and premalignant tumors, including breast fibrocystic disease and fibroadenomas, benign prostatic hyperplasia, colorectal adenomas, anaplastic astrocytomas, and benign meningiomas and leiomyomas, in general no telomerase activity was detected; however, it was found in malignant tumor stages . In this way, telomerase activity is associated with the acquisition of malignancy. The detection of telomerase activity at preneoplastic or benign growth stages may signify disease progression and be of diagnostic value. For example, telomerase activity has been found in some cases of benign prostate hyperplasia and of benign giant tumors of the bone - all tissues that may progress to malignant tumors. As shown by Hiyama et al. in breast cancer, telomerase provides a useful diagnostic tumor marker: Among samples obtained by fine-needle aspiration, 14 of 14 patients whose aspirates contained detectable telomerase activity, and who subsequently underwent surgery, were confirmed to have breast cancer. Certain tumor types, such as neuroblastoma, display a lower telomerase activity in early-stage cancers, whereas expression in late-stage cases is higher. Neuroblastomas of a special stage (stage IV), which had short telomeres and no or weak telomerase activity, tended to regress spontaneously —possible proof of a correlation between an enzyme activity too weak to remain in an immortal tumor status and a favorable outcome for the patient.

Another example of telomerase activity in cancer diagnosis and as a prognostic indicator of clinical outcome is the results found in gastric cancers. Hiyama et al. showed that the survival rate of patients with tumors with detectable telomerase activity in their study was shorter than that of those without telomerase activity. Although a reliable tumor marker, telomerase activity is not an all-or-none phenomenon. To understand the regulation of telomerase during tumorigenesis, Greider et al. analyzed the concentrations of telomerase RNA components and discussed the differential regulation of enzyme activity according to the concentration of the RNA component . Further prospective and retrospective clinical studies must be carried out to assess the validity of telomere dynamics and telomerase as a diagnostic or prognostic marker in many cancer types.

Up-Regulation of Telomere-Binding Proteins, TRF1, TRF2, and TIN2 is Related to Telomere Shortening during Human Multistep Hepatocarcinogenesis

The telomeric repeat-binding factor 1 (TRF1), TRF2, and the TRF1-interacting nuclear protein 2 (TIN2) are involved in telomere maintenance. We describe the regulation of expression of these genes along with their relationship to telomere length in hepatocarcinogenesis. The transcriptional expression of these genes, TRF1 protein, and telomere length was examined in 9 normal livers, 14 chronic hepatitis, 24 liver cirrhosis, 5 large regenerative nodules, 14 low-grade dysplastic nodules (DNs), 7 high-grade DNs, 10 DNs with hepatocellular carcinoma (HCC) foci, and 31 HCCs. The expression of TRF1, TRF2, TIN2 mRNA, and TRF1 protein was gradually increased according to the progression of hepatocarcinogenesis with a marked increase in highgrade DNs and DNs with HCC foci and a further increase in HCCs. There was a gradual shortening of telomere during hepatocarcinogenesis with a significant reduction in length in DNs. Most nodular lesions (52 of 67) had shorter telomeres than their adjacent chronic hepatitis or liver cirrhosis, and the telomere lengths were inversely correlated with the mRNA level of these genes (P ≤ 0.001). This was more evident in DNs and DNs with HCC foci. In conclusion, TRF1, TRF2, and TIN2 might be involved in multistep hepatocarcinogenesis by playing crucial roles in telomere shortening.

Telomerase as a target for anticancer therapy These findings suggest that reactivation of telomerase may be an obligate event in cell immortalization and in most instances of tumorigenesis. Any kind of inhibition of the enzyme should therefore lead to resumption of telomere shortening and might activate the cellular senescence pathway. The fact that telomerase is absent and not required in most human somatic tissues but is necessary for tumor growth should make this enzyme an ideal target for anticancer therapy. One of the greatest challenges in cancer therapy is to achieve a high therapeutic effect by maximizing the desired reactions and minimizing the undesired side-effects. Several conditions must be successfully fulfilled to achieve a beneficial antitumor effect in vivo: 1) Introduction of the antitumor agent into the majority and perhaps 100% of the tumor cells 2) The antitumor agent reaching the target 3) Tumor cell death 4) Acceptable toxicity to normal cells 5) Absence of a deleterious host immune response. One of the most hopeful approaches to achieving these goals is oligonucleotide-based gene therapy. The regulation of expression of genetic information by complementary pairing of sense and antisense nucleic acid strands has been termed ―antisense.‖ It is now possible to design antisense DNA oligonucleotides or antisense RNAs that can pair with and functionally inhibit the expression of genes in a sequence fashion. This high degree of specificity has made antisense constructs attractive candidates for therapeutic agents . Because the antisense sequences require chemical modifications to avoid destruction by nucleases and to form complexes for better delivery into the cell, peptide nucleic acids (PNAs) have been designed—with a charge-neutral, pseudo-peptide backbone of N-(2aminoethyl)glycine units instead of a negatively charged deoxyribosephosphate backbone . Recently, Norton et al. reported on PNAs that have a sequence complementary to the RNA component of human telomerase. They designed oligonucleotides that bind to the RNA molecule within the telomerase, which serves as the enzyme’s own internal telomere template. These molecules seem to be very specific and efficient inhibitors of telomerase in vitro. Further experiments with cell cultures and tumor models in mice will continue this path of investigation and could justify the optimism surrounding the telomerase hypothesis and its exploitation as a novel anti-cancer therapy.

Several important unanswered questions remain. A possible therapeutic approach of telomerase to cancer patients would appear to be less toxic than conventional chemotherapy, which affects all dividing cells and has undesirable side effects. However, some normal somatic cells are telomerase-positive at baseline: human hematopoietic progenitor cells, germ cells (progenitors of sperm and oocytes), and activated T-and Blymphocytes. Telomeric DNA in these cells would be lost during an ―antitelomerase therapy.‖ Perhaps this loss could be buffered, because of the longer telomeres in these cells than in cancer cells and because of the lower division rate relative to tumor cells. Another problem is in the variability of telomere lengths among tumors. Telomerase inhibition seems to be useful only in malignant cells with short telomeres; tumor cells with long telomeres would require a prolonged treatment, with possible toxic side-effects. One should also consider that alternative pathways besides telomerase may exist for regulating telomere length. Many other issues remain to be resolved. Even if the causal relationship were clear between continually shrinking telomeres and cellular senescence on the one side and unlimited proliferation of cells that have stabilized their telomeres’ length by the enzyme telomerase on the other, we still need to learn: Which genes encode telomerase components? Are telomeric RNA and proteins regulated at the transcriptional or posttranscriptional level? Does regulation of the RNA or of the protein components determine activity? Do internal cellular mechanisms exist to repress telomerase? Based on the present knowledge of telomeres and telomerase mechanisms, considerable efforts will have to be taken to answer these open questions and to make use of the new results for developing new diagnostic tools and therapeutic strategies.

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